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Characterization of Interaction Between Brevetoxinand Its Native Receptor and Identification of theRole of Brevetoxin in Karenia brevisWei ChenFlorida International University, [email protected]

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FLORIDA INTERNATIONAL UNIVERSITY

Miami, Florida

CHARACTERIZATION OF INTERACTION BETWEEN BREVETOXIN AND ITS

NATIVE RECEPTOR AND IDENTIFICATION OF THE ROLE OF BREVETOXIN

IN KARENIA BREVIS

A dissertation submitted in partial fulfillment of

the requirements for the degree of

DOCTOR OF PHILOSOPHY

in

CHEMISTRY

by

Wei Chen

2016

ii

To: Dean Michael R. Heithaus College of Arts, Sciences and Education This dissertation, written by Wei Chen, and entitled Characterization of Interaction Between Brevetoxin and Its Native Receptor and Identification of the Role of Brevetoxin in Karenia brevis, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved.

_______________________________________

Yuan Liu

_______________________________________ Xiaotang Wang

_______________________________________

Jose M. Eirin-Lopez

_______________________________________ Rudolf Jaffe

_______________________________________

Kathleen Rein, Major Professor

Date of Defense: November 7, 2016 The dissertation of Wei Chen is approved.

_______________________________________ Dean Michael R. Heithaus

College of Arts, Sciences and Education

_______________________________________ Andrés G. Gil

Vice President for Research and Economic Development and Dean of the University Graduate School

Florida International University, 2016

iii

© Copyright 2016 by Wei Chen

All rights reserved.

iv

DEDICATION

I dedicate this work to my parents, my grandparents, my boyfriend and his family for

their endless love and support. Without their understanding and encouragement, the

completion of this work would not have been possible.

v

ACKNOWLEDGMENTS

I am really grateful to the numerous people who help me a lot in the pursuit of this

Ph.D degree. Without their guidance and support the accomplishment of this

dissertation would not have been possible. It is my great pleasure to express my

gratitude to all of them in my humble acknowledgment

First and foremost, I would like to thank my major professor Dr. Kathleen Rein for

the opportunity to study and work in her group to pursue a doctoral degree. Without

her endless support, patience and supervision, my skill of writing and presenting

could not be improved during entire degree process. I do appreciate everything she

has done for encouraging me to overcome lots of obstructions during the past five

years.

I would also like to thank my other committee members, Dr. Yuan Liu, Dr. Xiaotang

Wang, Dr. Rudolf Jaffe and Dr. Jose M. Eirin-Lopez for their suggestion, guidance

and valuable time througout the years.

I would like to acknowledge all the faculty and staff members of the chemistry

department for the support, help and coordination. Big thanks to the dean’s office of

the College of Arts, Sciences and Education for travel funding.

I would like to thank my lab mates for their support and friendship. Because of you,

my five years are full with love and happiness. Special thanks to Li Liu, Ryan Cassell

Pengfei Sun and Freddy Rodriguez for their help in my study.

Finally, I wish to express my appreciation to those who have helped me in any respect

during my study at FIU, as well as expressing my apology that I could not mention

personally one by one.

vi

ABSTRACT OF THE DISSERTATION

CHARACTERIZATION OF INTERACTION BETWEEN BREVETOXIN AND ITS

NATIVE RECEPTOR AND IDENTIFICATION THE ROLE OF BREVETOXIN IN

KARENIA BREVIS by

Wei Chen

Florida International University, 2016

Miami, Florida

Professor Kathleen Rein, Major Professor

Algae are important to marine and fresh-water ecosystems. However, some species

of algae are harmful or even toxic. They can consume oxygen or block sunlight that

is essential for other organisms to live. Indeed, some algae blooms can produce

toxins that damage the health of the environment, plants, animals, and humans.

Harmful algal blooms (HABs) which are often more green, brown, or dark-colored

than red have spread along the coastlines and in the surface waters of the United

States. Therefore, scientists are making great efforts to study HABs in order to

maintain human and ecosystem health.

Karenia brevis, the major harmful algal bloom dinoflagellate of the Gulf of Mexico,

plays a destructive role in the region. Karenia brevis, responsible for Florida red tide,

is the principle HAB dinoflagellate in the Gulf of Mexico. K. brevis blooms can

produce brevetoxin: ladder-shaped polyether (LSP) compounds, which can lead to

adverse human health effects, like reduced respiratory function through inhalation

exposure, or neurotoxic shellfish poisoning through consumption of contaminated

shellfish. The poisoning has been attributed to their affinity for voltage-sensitive

sodium ion channels causing channel opening and depolarization of excitable cell

vii

membranes. Conservative estimate suggests that the economic impact from all

harmful algal bloom events in the United States is at least $82 million/year. The

public health costs occupy $37 million alone.

The study presented herein utilized fluorescent and photolabile brevetoxin probes to

demonstrate that brevetoxin localizes in the chloroplast of K. brevis where it binds

to light harvest complex II (LHC II) and thioredoxin (Trx). It had been discovered

that the TrxR/Trx system was inhibited by brevetoxin-2 (PbTx-2) with an IC50 of 25

µM. The mechanism of the inhibition was discussed in this work. The research also

revealed that the K. brevis high-toxic and low-toxic strains have a significant

difference in their ability, not only to produce brevetoxin, but also to perform NPQ

and in the production of ROS. I compared and contrasted various metabolic and

biochemical parameters in two strains of K. brevis which had a ten-fold difference in

toxin content. The work could shed light on the physiological role that brevetoxin

fills for K. brevis and may contribute to understanding the effect of ladder-shaped

polyether compounds on both marine animals and exposed humans and shall inform

improved treatments for brevetoxicosis.

viii

TABLE OF CONTENTS

CHAPTER PAGE

Chapter 1 Introduction ................................................................................................... 1 1.1 Harmful algal blooms (HABs) ................................................................................ 1 1.2 Karenia brevis ( K. brevis ) ..................................................................................... 4

1.2.1 The historical records of K. brevis ............................................................... 5 1.2.2 The historical effects of K. brevis ................................................................ 6

1.3 Brevetoxins and related ladder-shaped polyether (LSP) compounds ...................... 7 1.4 Biological activities of LSPs ................................................................................. 12

1.4.1 Biological activities of brevetoxin on animal ............................................ 12 1.4.2 Biological effects of brevetoxin on human ................................................ 15 1.4.3 The effect of brevetoxin on cellular process .............................................. 16

1.5 LSP compounds bind to transmembrane protein .................................................. 18 1.6 The interaction between PbTxs VGSC at the molecular level .............................. 20 1.7 The effect of brevetoxin on economy .................................................................... 23 1.8 Brevetoxins play an important role in K. brevis, but it is still elusive .................. 24 1.9 The significance of studying the dinoflagellates ................................................... 26 1.10 Research objectives ............................................................................................. 26

Chapter 2 Localization of PbTxs to a subcellular organelle and identification of a target protein from K. brevis ........................................................................................ 28

2.1 Localize PbTxs to one or more organelles or cellular structures in K.brevis ........ 28 2.1.1 Objective .................................................................................................... 28 2.1.2 Introduction ................................................................................................ 28 2.1.3 Results and discussion ............................................................................... 31

2.2 Isolation of a target protein from K. brevis as PbTxs natural receptor .................. 33 2.2.1 Objective .................................................................................................... 33 2.2.2 Introduction ................................................................................................ 33 2.2.3 Results and Discussions ............................................................................. 36 2.2.4 Conclusion ................................................................................................. 42

2.3 Materials and Method ............................................................................................ 44 2.3.1 Fluorescent probe experiment .................................................................... 44 2.3.2 Photoaffinity probe experiment ................................................................. 45

Chapter 3 The effects of brevetoxins on the thioredoxin reductase/thioredoxin system and related enzymes ..................................................................................................... 50

3.1 Objective ............................................................................................................... 50 3.2 Introduction ........................................................................................................... 50 3.3 Results ................................................................................................................... 59

3.3.1 Modified papain assay ............................................................................... 59 3.3.2 Papain assay ............................................................................................... 60 3.3.3 Fluorescent assay for Trx/TrxR system ..................................................... 64 3.3.4 Ellman’s assay ........................................................................................... 66 3.3.5 Adduct formation between PbTx-2 and selenocysteine ............................. 72 3.3.6 Adduct formation between PbTx-2 and TrxR ........................................... 73 3.3.7 Reaction of TrxR with Sel-green probe ..................................................... 75 3.3.8 Grx, GPx fluorescent assay ........................................................................ 76

ix

3.4 Conclusion ............................................................................................................. 78 3.5 Materials and Methods .......................................................................................... 80

3.5.1 Modified papain assay ............................................................................... 80 3.5.2 Papain assay ............................................................................................... 81 3.5.3 Fluorescent assay for Trx/TrxR system ..................................................... 82 3.5.4 Ellman’s assay ........................................................................................... 83 3.5.5 The preparation of selenocysteine -PbTx-2 adduct ................................... 84 3.5.6 The preparation of TrxR-PbTx-2 adduct. .................................................. 84 3.5.7 Reaction of Sel-green probe with TrxR ..................................................... 85 3.5.8 Fluorescent assay for Grx and GPx ........................................................... 86

Chapter 4 Comparison of biochemical parameters in high toxin and low toxin producing strains of K. brevis ....................................................................................... 89

4.1 Objective ................................................................................................................. 89 4.2 Introduction ............................................................................................................. 89 4.3 Results and discussion ............................................................................................. 98 4.3.1 The antioxidants in two strains of K. brevis .............................................. 99

4.3.2 Redox state of protein and non-protein thiols in low vs high toxin cultures of K. brevis .......................................................................................... 102

4.3.3 The reaction of Sel-green probe in two K. brevis strains. ........................ 105 4.3.4 The activity of Trx and Grx in two K. brevis strains ............................... 106

4.4 Conclusion ............................................................................................................. 107 4.5 Materials and Methods .......................................................................................... 110

4.5.1 Culture methods ....................................................................................... 110 4.5.2 Preparation of K. brevis homogenate ....................................................... 110 4.5.3 ABTS assay .............................................................................................. 110 4.5.4 Modified papain assay ............................................................................. 111 4.5.5 Ellman’s assay ......................................................................................... 112 4.5.6 Sel- green reacts with homogenate of tow K. brevis strains .................... 113 4.5.7 Thioredoxin activity in homogenate of two strains K. brevis .................. 114 4.5.8 Grx activity in homogenate of two strains K. brevis ............................... 115

Chapter 5 Summary and future work ........................................................................... 116

5.1 Summary ............................................................................................................... 116 5.2 Future work ........................................................................................................... 117

5.2.1 Identify all biological receptors for brevetoxin in K. brevis and localize the binding site on native receptors ................................................................. 117 5.2.2 Analysis of the essential proteins, carotenoids and lipids for NPQ in low vs high toxin K. brevis ............................................................................... 119 5.2.3 Determination the role of brevetoxin in two K. brevis strains ................ 120

References .................................................................................................................... 121

VITA ............................................................................................................................ 144

x

LIST OF TABLE

TABLE PAGE

Table 1 Human poisoning syndromes caused by HAB ................................................. 4

Table 2 The peptide sequence of 27 kD band and the proteins with matching sequences. .................................................................................................................... 37 Table 3 The intensity of all bands on SDS-PAGE gel ................................................. 41

Table 4 The peptide sequence of the 12 kDa band the proteins with matching sequences ..................................................................................................................... 41 Table 5 The motifs in thioredoxin , glutaredoxin, PDI and DsbA .............................. 51

Table 6 Relative V0 for the assays shown above in Figure 24,26 and 27 .................... 63

Table 7 The inhibition % for the assays shown above in Fig 24,26 and 27 ................ 64

Table 8 The predicted Se isotopic ratio ....................................................................... 73

Table 9 The major ROS molecules and their metabolism ........................................... 90

Table 10 The alignments of TrxR in K. brevis, Homo sapiens and Emiliania huxleyi ......................................................................................................... 99 Table 11 Comparison of NPQ and ROS in two K. brevis strains .............................. 108

Table 12 Comparison of various biochemical parameters in two K. brevis strains at 19th days ................................................................................................................. 108

xi

LIST OF FIGURES

FIGURE PAGE

Figure 1 Karenia brevis ................................................................................................. 5

Figure 2 The structure of brevetoxin A .......................................................................... 8

Figure 3 The structure of brevetoxin B .......................................................................... 8

Figure 4 The structure of ciguatoxin B .......................................................................... 8

Figure 5 The structure of yessotoxin ............................................................................. 8

Figure 6 Structures of small polyether ladder toxins produced by ................................ 9

Figure 7 Synthesis of fluorescent brevetoxin probe .................................................... 31

Figure 8 The photograph of localization of brevetoxin in K. brevis live cells ............ 32

Figure 9 The structure of okadaic acid ........................................................................ 33

Figure 10 The most reliable and common high efficiency labeling of target proteins in photoaffinity probe ..................................................................................... 34 Figure 11 The synthesis of brevetoxin photoaffinity probe ......................................... 36

Figure 12 SDS-PAGE of protein fractions from photoaffinity experiments ............... 37

Figure 13 Xanthophyll cycle ........................................................................................ 39

Figure 14 Spinach homogenate was incubated with the photoaffinity probe in different pH buffer ....................................................................................................... 41 Figure 15 Trx catalyzes the reaction as a nucleophilic substitution reaction (SN2) ..... 52

Figure 16 Electron flow of E. coli TrxR/Trx system ................................................... 53

Figure 17 Reaction mechanism of TGR ...................................................................... 55

Figure 18 Electron flow of mammalian TrxR/Trx system ........................................... 56

Figure 19 The Trx/TrxR system plays a key role in biochemical processes associated with many human diseases ......................................................................... 57 Figure 20 Electron flow of mammalian GSH/GSSG system ....................................... 58

Figure 21 The redox cycle of GPx coupled with mammalian GSH/GSSG system ..... 59

xii

Figure 22 The equation of modified papain assay ....................................................... 59

Figure 23 The principle of modified papain assay to determine Trx activity .............. 60

Figure 24 The modified papain assay with / without PbTx-2 ...................................... 61

Figure 25 The modified papain assay with / without PbTx-2 but without cystamine. 62

Figure 26 The modified papain assay with / without PbTx-3 with cystamine ............ 62

Figure 27 The papain assay with / without brevetoxin ................................................ 63

Figure 28 The reaction in sensitive fluorescent assay for Trx/TrxR system ............... 64

Figure 29 The effect of PbTx-2 on Trx/TrxR system .................................................. 65

Figure 30 The effect of PbTx-3 on Trx/TrxR system .................................................. 66

Figure 31 The reduction of Ellman’s reagent .............................................................. 66

Figure 32 The effect of PbTx-2 on the Ellman’s assay ............................................... 67

Figure 33 The electron flow in C-terminal reaction center in mammalian TrxR ........ 68

Figure 34 The Michael acceptors demonstrated to inhibit TrxR ................................. 69

Figure 35 The hypothesized explanation for the observation in fluorescent Trx/TrxR assay with PbTx-2 ....................................................................................... 70 Figure 36 The hypothesized explanation for the observation in DTNB assay with TrxR and PbTx-2 ......................................................................................................... 70 Figure 37 The effect of truncated recombinant human ................................................ 71

Figure 38 The effect of PbTx-3 on DTNB reduction by mammalian TrxR ................ 72

Figure 39 The mass spectrum of adduct formation between PbTx-2 with selenocysteine .............................................................................................................. 73 Figure 40 MS/MS analysis of the peptides from rat liver TrxR .................................. 74

Figure 41 MS/MS analysis of the peptides from the adduct between PbTx-2 and TrxR ...................................................................................................................... 74 Figure 42 Reaction of Sel-Green probe with selenocysteine ....................................... 75

Figure 43 Release of fluorescent reporter from Sel-green probe upon incubation with TrxR in the presence and absence of PbTx-2 compared to standards of L-selenocysteine .............................................................................................................. 76

xiii

Figure 44 The principle of sensitive fluorescent assay for activity of Grx .................. 77

Figure 45 The reaction in sensitive fluorescent assay for GPx system ........................ 77

Figure 46 The effect of Grx activity by PbTx-2. ......................................................... 77

Figure 47 The effect of GPx activity by PbTx-2 ......................................................... 78

Figure 48 The common pathways to produce ROS ..................................................... 93

Figure 49 Major physiological function of the mammalian thioredoxin system ......... 95

Figure 50 The structure of carboxy-H2DCFDA ........................................................... 96

Figure 51 The ROS production in two strains of K. brevis ......................................... 96

Figure 52 NPQs for the low and high toxic algal cultures to the pulses of four different excitation wavelengths .................................................................................. 97 Figure 53 The reaction in ABTS assay ...................................................................... 100

Figure 54 The antioxidant capacity in two strains of K. brevis ................................. 101

Figure 55 The total amount of ascorbic acid in two stains of K. brevis .................... 101

Figure 56 The detection of thiols in K. brevis homogenate by modified papain assay ............................................................................................................... 103 Figure 57 The concentration of cells over the lifetime of high toxin and low toxic strains ......................................................................................................... 103 Figure 58 The redox state of high toxic and low toxic strains. .................................. 105

Figure 59 The totally glutathione in two strains of K. brevis and the ratio of native reduced glutathione to total glutathione in two strains of K. brevis .......................... 105 Figure 60 The fluorescence is released by the reaction of sel-green probe with thiol groups and selenol groups in K. brevis homogenate ......................................... 106 Figure 61 Using the fluorescent Trx / TrxR system kit detects the Trx activity in K. brevis homogenate ................................................................................................. 107 Figure 62 Using the fluorescent Grx kit detects the Grx activity in K. brevis homogenate ................................................................................................................ 107 Figure 63 Spinach homogenate was incubated with photoaffinity probe in PBS for 1 hour .................................................................................................................... 118

xiv

Figure 64 A-ring derivative from PbTx-2 or PbTx-3 A-ring diol ............................. 118

xv

LIST OF ABBREVIATIONS

AAO ascorbic acid oxidase

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid

Abs absorbance

ASP amnesic shellfish poisoning

ATP adenosine triphosphate

AZ tetrafluorophenyl azides

AZP azaspiracid poisoning

BP benzophenone

BSA bovine serum albumin

CFP ciguatera fish poisoning

CSD Cambridge Structural Database

Cys cysteine

Chl chlorophyll

1Chl* singlet chlorophyll

3Chl* triplet chlorophyll

DAPI 4, 6-diamidino-2-phenylin-dole)

D dinoxanthin

DD diadinoxanthin

DIA trifluoromethylphenyl diazirines

DMF dimethylfomamide

DNase deoxyribonuclease

DNA deoxyribonucleic acid

DSP diarrhefic shellfish poisoning

DTNB 5, 5’-dithio-bis-(2-nitrobenzoic acid)

xvi

DTT dithiothreitol

E. coli Emilliania huxlei

EDTA ethylenediaminetetraacetic acid

FAD flavin adenine dinucleotide

FITC fluorescein isothiocyanate

FT-ICR fourier transform ion cyclotron resonance mass spectrometer

FTR ferredoxin thioredoxin reductase

GpA glycophorin A

Gpx glutathione peroxidase

GR glutathione reductase

Grx glutaredoxin

GSH glutathione

GSSG glutathione disulfide

H2O2 hydrogen peroxide

hTrxR human thioredoxin reductase

IAC iodoacetamide

ICW Intracoastal Waterway

IC50 half maximal inhibitory concentration

IPCC Intergovernmental Panel on Climate Change

K. brevis K. brevis

kDa kilodalton

Ki the concentration to produce half maximum inhibition

Km the substrate concentration to produce half maxium

velocity

xvii

LipDH lipoarmide dehydrogenase

LSP ladder shape polyether

mRNA messenger ribonucleic acid

MW molecular weight

NADPH nicotinamide adenine dinucleotide phosphate

NCBI National Center for Biotechnology information

NMR nuclear magentic resonance spectroscopy

NO nitric oxide

NPQ non-photochemical quenching

NSP neurotoxic shellfish poisoning

NTRC NADPH dependent thioredoxin reductase

OA okadaic acid

O2- superoxide anion

. OH hydroxyl radical

PAL photoaffinity labeling

PAM pulsed amplitude modulation

PBS phosphate buffered saline

PbTx brevetoxin

PDI protein disulfide isomerase

PE polyether

Prx peroxiredoxins

PSP paralytic shellfish poisoning

ROS reactive oxygen species

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

xviii

Sec selenocysteine

SN2 nucleophilic substitution reaction

SNAr nucleophilic aromatic substitution

SOD superoxide dismutase

SPR surface plasmon resonance

TCEP tris (2-carboxyethyl) phosphine hydrochloride

TEAC trolox equivalent antioxidant capacity

TGR thioredoxin glutathione reductase

Tris Tris(hydroxymethyl)aminomethane

TNB 2-nitro-5-thiobenzoic acid

Trx thioredoxin

TrxR thioredoxin reductase

VDE violaxanthin de-epoxidase

VGSC voltage-gated sodium channel

YTX yessotoxin

ZE zeaxanthin epoxidase

2-Cys Prxs 2-cysteine peroxiredoxins

β bata

α alpha

0C degress Celsius

g gram

hr hour

l liter

µ micro

λ lamdba

xix

m milli

mol mole

min minute

% percentage

1

Chapter 1 Introduction

1.1 Harmful algal blooms (HABs)

Microscopic algae, or phytoplankton are common in coastal waters. Although

phytoplankton are small, they can grow explosively under certain conditions that are

not yet well understood, creating a phenomenon commonly known as algae blooms.

Algal blooms may occur in freshwater as well as in marine environments. During a

bloom, algae may become concentrated enough to turn the water red, brown or black.

These blooms are often referred to as red tides.

The algae reproduce rapidly when the nutrient levels increase above the normal

conditions. An intense bloom can cause harmful impacts on marine ecosystems. It can

consume oxygen or block sunlight that is essential for the survival of other organisms.

As a result, the concentrated algal blooms, which spread across the surface of the

ocean or freshwater, can inhibit the growth of other species (Baden, Bourdelais et al.

2005). Algal blooms may or may not occur seasonally, are often localized to specific

geographic regions, and may be triggered or exacerbated by environmental factors

(Baden, Bourdelais et al. 2005). These may be related to changes in species of algae’s

composition or other environmental cues. It has been hypothesized that eutrophication,

nutrient sources and some natural processes contribute to the occurrence of harmful

algae bloom, such as river runoff, pollution, changes in the temperature of the oceans

and so on. These all have been associated with blooms (Baden, Bourdelais et al. 2005).

Because of the nutrient rich environments, coastal regions are most severely impacted

by marine blooms. It has been suggested that blooms have increased in frequency and

duration along the coastlines and in the surface waters of the United States as a result

2

of human activities (Fleming, Backer et al. 2005). Many of the nutrients which

support algal growth may be found in certain fertilizers which are discharged into the

water as agricultural waste (Stumpf, Culver et al. 2003). Increased nitrogen (N) and

phosphorus (P) input resulting from human activities can stimulate bloom growth

along the coast (Harrison, Caraco et al. 2005, Seitzinger, Harrison et al. 2005,

Burkholder, Dickey et al. 2006, Bricker, Longstaff et al. 2008). In some cases, the

connections between algal blooms and eutrophication are clear and well documented.

For example, in the 1970s a reduction in red tide, which was proportional to mandated

pollution reductions, was observed in the Inland Sea of Japan for a decade (Takahashi,

Kawamura et al. 2009). Also, an increase in nutrient input in the northwestern Black

Sea in the 1980s, is correlated with an increase in algal blooms during the subsequent

two decades (Bodeanu, Moncheva et al. 1998). Over the past several decades, red tide

has occurred almost annually along the Gulf Coast of Florida, has spread along the

Texas and eastern Florida coasts, and occasionally along the coasts of Louisiana,

Mississippi, and Alabama (Thyng, Hetland et al. 2013). It is indicated that

eutrophication is responsible for the increasing of the red tide in Gulf of Florida

(Heisler, Glibert et al. 2008). However, the mechanisms of the stimulation of algal

bloom by nutrients are not always clear and remain controversial.

In addition to eutrophication, climate change is among the suspected factors for the

increased frequency of blooms. Global warming has led to the temperature rising on

the surface of ocean. Recently, the Intergovernmental Panel on Climate Change

(IPCC) verified that over the past 40 years, the ocean absorbed 90 percent of earth’s

net energy, resulting in nearly 4 oC increase in the ocean (Collins, Knutti et al. 2013).

Warmer water provides blooms a favorable environment to grow. As a result, a range

3

expansion of algal bloom has been observed under warm weather conditions.

However, debate on the ultimate cause of range expansions is ongoing (Smayda 2007,

Hallegraeff 2010).

Although algae are essential to marine and fresh-water ecosystems, some species of

algae are harmful or even toxic. Some algae can produce toxins that damage the

health of the environment, plants, animals, and humans. These algal blooms, which

can cause environmental or public health threats, are referred to as harmful algal

blooms (HABs) (Hallegraeff 1993). Some HABs are associated with the production

of algal toxins. Toxins are a form of secondary metabolite; or organic compounds that

are not essential to the normal growth or development of an organism. Secondary

metabolites are typically produced by one species or a few closely related species and

are generally believed to convey a selective advantage to the producing organism.

Toxins pose a serious threat to other species sharing the aquatic environment with the

algae. Animals, who consume contaminated seafood, sea grass, and contaminated

seawater or inhale contaminated aerosols can suffer severe poisoning often resulting

in mass mortalities of marine animals. For example, the historical records have shown

that Florida red tide bloom can cause massive mortalities of at least 100 species of

marine animals, such as shellfish, sea birds, manatees and dolphins (Martin and

Martin 1976). A number of human poisoning syndromes are caused by ingestion of

HAB related toxins in contaminated seafood (Table 1) (Anderson, Moore et al. 2015),

such as paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP),

amnesic shellfish poisoning (ASP), ciguatera fish poisoning (CFP) and azaspiracid

poisoning (AZP), which are caused by HAB related toxins (Anderson, Moore et al.

2015). An alternate exposure route resulting from human activities or wave action can

4

release toxins into ambient water or air. Algal toxins are generally very stable

molecules and can persist in the environment unchanged for long periods.

Table 1 Human poisoning syndromes caused by HAB

SYNDROME SPECIES TOXIN Symptoms

Paralytic Shellfish Poisoning (PSP)

Alexandrium spp. Pyrodinium spp.

Gymnodiunium spp. Saxitoxin Numbness, vomiting,

diarrhea and others.

Neurotoxic Shellfish Poisoning

(NSP) Karenia spp Brevetoxin Nausea, vomiting and

muscle weakness

Amnesic Shellfish poisoning (ASP)

Pseudo-nitzschia spp. Nitzschia navis-varingica Domoic Acid

Nausea, vomiting, headache, dizziness and

others l

Ciguatera Fish Poisoning (CFP) Gambierdiscus spp. Ciguatoxins Nausea, vomiting,

diarrhea, rash and others

Azaspiracid poisoning (AZP) Azadinium spp. Azaspiracid

Nausea, vomiting, abdominal cramps and

others l

Diarrhetic shellfish poisoning (DSP)

Dinophysis spp. Prorocentrum spp. Okadaic acid

Nausea, vomiting abdominal cramps and

others

1.2 Karenia brevis ( K. brevis )

Karenia brevis, formerly known as Gymnodinium breve and Ptychodiscus brevis, is

the major HAB forming organism in the Gulf of Mexico. Karenia brevis is a

microscopic, single-celled, photosynthetic dinoflagellate which is responsible for the

Florida “red tide”. Karenia brevis cell size is usually between 20 and 40 nm in

diameter. The organism has cellulose plates, called theca, surrounding the cell as

protection, which creates the boundary for the cell. On the cellulose plates there are

two flagellum located in two grooves called the transverse and longitudinal grooves.

Two flagella allow K. brevis to move through water with a spinning motion. Usually,

one of flagellum wraps around the cell in the transverse groove whereares the other

5

one extends on the longitudinal groove. Both the theca and flagella are visible in

Figure. 1. The nucleus is located in the lower part of the cell. Usually, becuase of the

presence of chloroplasts, K. brevis is yellow-green in color. Karenia brevis is an

obligate autotroph with facultative heterotrophy. It can gain energy from some

organic molecules and utilizes inorganic molecules such as nitrogen and phosphorus.

It also can produce energy from photosynthesis (Hitchcock, Kirkpatrick et al. 2010).

Figure 1 Karenia brevis. Image courtesy of Florida Fish and Wildlife Conservation Commission

Light is an essential requirement for K. breivs, thus it cannot survive at depths below

200 feet. Normally K. brevis is found on the surface of the water. The optimal

temperature range for K. brevis is 22-28 degrees Celsius. In addition, K. breivs can

live in a salinity range of 25-45 ppt. Under the appropriate conditions including

sufficient light and nutrients, K. brevis can concentrate from 100,000 to 250,000 cells/

liter within 2 months (Tester and Steidinger 1997). Blooms of K. brevis may also

provide a significant source of fixed carbon thorough photosynthesis (Vargo, Heil et

al. 2002).

1.2.1 The historical records of K. brevis

Karenia brevis blooms were first recorded centuries ago. According to historical

records, a red tide was first mentioned and documented in Gulf of Mexico in 1658

(Magaña, Contreras et al. 2003). It has been documented on the Florida west coast

6

since the 1800s. In the past decade, the Florida red tide blooms have been widespread

in the Gulf of Mexico, especially from Clearwater to Sanibel Island (Backer,

Kirkpatrick et al. 2005). Most commonly, K. brevis blooms can last more than two

months in regions of the Gulf of Mexico including: the west Florida shelf, the

Campeche Bay between Rio Ciatzacoalcos and Rio Grijalva, and Texas coast between

Port Arthur and Galveston Bay (Tester and Steidinger 1997). A bloom which

occurred in 2005 persisted for nearly one year (Hoagland, Jin et al. 2014).

Blooms of K. breveis result in detrimental effects to marine life and local economies

and as people living, working, and visiting the affected coastal areas (Backer,

Kirkpatrick et al. 2005). It is documented that cell densities as low as 103 cell/L will

cause respiratory irritation, whereas a concentration of more than 5 x 103 cells/L can

induce neurotoxic effects. Massive fish kills can be caused by densities over 105

cell/L, and cell densities over 106 cell/L result in water discoloration (Backer, Fleming

et al. 2003). An offical government record described the first reported fish kill in the

Gulf of Mexico in 1792 in the city of Veracruz, Mexico (Magaña, Contreras et al.

2003). It was reported that multitudes of dead fish washed onto the beaches of

Veracruz. The document also recored that same phenomenon occurred repeatedly in

that port.

1.2.2 The historical effects of K. brevis

It has been proposed that there are different effects which led to the massive fish and

marine animals killed by Florida red tide such as oxygen deprivation and air-born

aerosols.

7

Oxygen deprivation: Biochemical decomposition begins when the red tide organisms

die, which can cause oxygen level to decrease rapidly. In July 1971, a large spread of

red tide in Tampa Bay completely devastated the benthic ecosystem in the Bay.

Oxygen deprivation, rather than toxin, was one of the considerable contributors to the

death of creatures such as turtles, barnacles, birds and sponges (Simon and Dauer

1972).

Air-born aerosols: In the late 1800s massive fish kills along with human respiratory

irritation and dry cough in Veracruz, Mexico were reported when a K. brevis bloom

reached high concentration (Tester and Steidinger 1997, Magaña, Contreras et al.

2003). It is believed that highly concentrated K. brevis blooms played an essential role

in the poisoning of marine life. In 1996, at least 149 manatees died along the

southwest coast of Florida. According to lung pathology, a K. brevis produced toxin

was detected in the lung (Flewelling, Naar et al. 2005). It is highly likely that the

toxin had been inhaled by the manatees.

1.3 Brevetoxins and related ladder-shaped polyether (LSP) compounds

Brevetoxin are the parent of a small class of compounds called polyether ladders

which are classified as polyketides according to their biogenic origin (Lee, Qin et al.

1989). The best known LSP are brevetoxin A (PbTx-A) (Figure 2), brevetoxin B

(PbTx-B) (Figure 3), ciguatoxin (Figure 4) and yessotoxin (Figure 5).

8

Figure 2 The structure of brevetoxin A

Figure 3 The structure of brevetoxin B

Figure 4 The structure of ciguatoxin B

Figure 5 The structure of yessotoxin

9

Inspection of these structures reveals numerous common features: these compounds

consist of trans fused polyether rings with oxygen atoms that alternate from the

bottom to top of the molecule (Rein and Borrone 1999). In the past decades, there are

not only numerous polycyclic ethers were isolated from blooms or laboratory culture

of K. brevis, such as brevisamide, hemibrevetoxin and brevisin (Figure 6) (Satake,

Campbell et al. 2009) and brevenal (Bourdelais, Campbell et al. 2004).

Figure 6 Structures of small polyether ladder toxins produced by dinoflagellates Brevisin is a polycyclic ether that is consisted of two separate fused polyether rings

linked with a methylene group. Brevisin and brevenal are both natural competitors of

brevetoxin for its binding site (Bourdelais, Campbell et al. 2004, Satake, Campbell et

al. 2009). Those polyether compounds hold great interest among many scientific

groups because of their unique structure and enigmatic biosynthetic origin. However,

the first challenge was characterization of polyether compounds. The molecular

structure of brevetoxin B was first determined by Nakanishi’s group in 1981 (Lin,

Risk et al. 1981). The absolute stereochemistry of brevetoxin B was elucidated by X-

10

ray crystallography (Shimizu, Bando et al. 1986). The determination of the first

polyether ladder structure by X-ray crystallography simplified the subsequent

structure elucidation of other polyether ladders. The structure of brevisin was

determined by MS and 2D NMR (Satake, Campbell et al. 2009). The structure of the

even more complex polyether ladder, ciguatoxinn, which is isolated from

dinoflagellate G. toxicus was elucidated by NMR (Murata, Legrand et al. 1989,

Murata, Legrand et al. 1990).

The biosynthetic pathways for these natural polyether ladder products remain a puzzle.

As a result of the lack of enzymes or gene sequence for biosynthetic pathways, stable

isotope labeling experiments and structural features were used to develop the

biosynthetic principles. In 1985, Nakanishi proposed the currently held hypothesis for

the biosynthetic pathway of brevetoxin. It was hypothesized that these polyether

ladder compounds arise from the cyclization of a polyepoxide precursor (Nakanishi

1985).

Numerous individual brevetoxins (PbTxs) have been isolated and characterized from

K. brevis bloom as well as laboratory culture. Taken together, PbTxs are lipid-soluble

polyether compounds with molecular weights around 1000 D (Backer, Fleming et al.

2003; Backer, Kirkpatrick et al. 2005). There are two principal types of PbTxs that

differ according to the carbon skeleton; PbTx-A and PbTx-B. A flexible backbone of

10 fused polyether rings makes up PbTx-A which is the minor, but most toxic

congener. PbTx-B, the most abundant of the two, is comprised of a rigid backbone of

11 polyether rings (Nakanishi 1985, Baden 1989). Almost a dozen derivatives of

PbTx-A and PbTx-B occur naturally. Principal A-type brevetoxin include PbTx-1,

11

PbTx-7 and PbTx-10, while PbTx-2, PbTx-3, PbTx-8 and PbTx-9 belong to B-types

brevetoxins. Others, such as PbTx-8, a chloromethyl ketone derivative of PbTx-2,

appears to be an artifact of chloroform extraction of PbTx-2 (Nakanishi 1985, Matile,

Berova et al. 1996, Baden, Bourdelais et al. 2005, Murrell and Gibson 2009); In the

Florida red tide PbTx-2 is the major brevetoxin congener in bloom water, while

PbTx-3 is contained as greatest amount in aerosols (Pierce, Henry et al. 2005).

Furthermore, some polar brevetoxin derivatives were isolated from K. brevis cultures

and natural blooms as well. PbTx-1 and PbTx-2 both possess a lactone functionality

in the A- ring and share a common, rigid region in the terminal four rings with a side

chain allowing modification (Rein, Baden et al. 1994, Rein, Lynn et al. 1994). All

natural brevetoxins and derivatives have these features. However, in fact no derivative

is more toxic than PbTx-1 or PbTx-2 (Baden, Bourdelais et al. 2005). As identified by

LC/MS, some brevetoxin derivatives were confirmed as open A-ring of brevetoxin

PbTx-1, PbTx-7, PbTx-2 and PbTx-3. Some are oxidized from PbTx-1 and PbTx-2

(Abraham, Plakas et al. 2006).

The synthesis of LSPs remains a significant challenge. After twelve years of effort,

the first total synthesis of brevetoxin B was reported in 1995 (Nicolaou 1996,

Nicolaou, Yang et al. 1998). In the first generation strategy, hydroxyl epoxide

cyclization and the intramolecular conjugate addition were used as key reactions to

form the fused tetrahydropyran ring systems (Ring ABC, ring FG and ring IJK).

However, the step to thionate and bridge the last remaining bond required for

completion of the brevetoxin B skeleton was unsuccessful (Nicolaou, Hwang et al.

1995). It failed to construct the fused oxepane rings in brevetoxin B simultaneously.

Thus, stepwise bis (oxepane) synthesis approach was used to display the first strategy.

12

However, the goal to fuse additional rings onto the DEFG ring system is still elusive

(Nicolaou, Theodorakis et al. 1995). Finally, in his third strategy he successfully

formed the ring ABCDEF and ring IJK, leaving the didehydrooxocane ring H as final

ring to construct. In total, this process consists of 123 steps (Nicolaou, Rutjes et al.

1995). Nowadays, several groups have synthesized brevetoxin successfully. In 2004,

brevetoxin B was synthesized in 90 steps (Stephen and Kettle 1997, Matsuo,

Kawamura et al. 2004).

1.4 Biological activities of LSPs

The LSP compounds are responsible for a variety of some poisonings. Maitotoxin and

ciguatoxin both isolated from G. toxicus are associated with the CFP symptoms such

as chills, sweating, neurologic dysfunction and generalized weakness lasting from 10

min to 30 hours from consuming contained fish or food (Levine 1995). Yessotoxin

(YTX) which is produced by the dinoflagellate Protoceratium reticulatum,

accumulates in the gland of Japanese scallops (Murata, Kumagai et al. 1987, Satake,

MacKenzie et al. 1997). Yessotoxin associated lethality occurs through acute heart

disease. According to histopathology, mice exposed to yessotoxin, exhibited

intercytoplasmic edema (Satake, MacKenzie et al. 1997). Thus, cardiac muscle cells

are affected by yessotoxin.

1.4.1 Biological activities of brevetoxin on animal

Brevetoxins can persist in the ecosystem for up to eight months after the absence of

K. brevis blooms. For an instance, in 2007 K. brevis was first observed in early

September near Jacksonville, FL. Subsequently, it expands through the east coast of

13

Florida, including the Indian River Lagoon and the Atlantic Intracoastal Waterway

(ICW) (Walsh, Weisberg et al. 2009). The maximum concentration of K. brevis was

up to 106 cell / L in October. The bloom terminated in December 2007. However,

brevetoxin had been detected in sediments and seagrass epiphytes in July and August

2008 in ICW. The highest concentration of brevetoxin in sediments has been found

near Patrick Air Force Base at 89 ng per gram of dry sediment (Hitchcock,

Fourqurean et al. 2012). Average concentration is < 20 ng per gram of dry sediment

during non-bloom periods (Flewelling 2008). At the same time, brevetoxins were

detected in six of nine seagrass beds sampled at the south of the Mosquito Lagoon at

concentration of 6-18 ng per gram of dry epiphytes (Hitchcock, Fourqurean et al.

2012). In general, brevetoxins can persist almost one year in either seagrass epiphytes

or sediments after the termination of a bloom.

Since brevetoxins remain in seagrass and sediments for several months following

bloom termination, it seems highly likely that marine life can consume the brevetoxin

from contaminated seagrass or sediments. Subsequently, brevetoxins may be

transported through the food chain. In the other words, blooms of brevetoxin-

producing K. brevis have been associated with the mortality of fishery resources as

well as the death of marine mammals such as the endangered Florida manatees,

dolphins, turtles and shorebirds. The major route of exposure related to the deaths of

dolphins, manatees, turtles and seabirds is ingestion of PbTxs. For instance, some

shorebirds such as sanderlings (Calidris alba) and ruddy turnstones (Arenaria

interpres) scavenged dead fish deposited onshore during a K. brevis bloom. It is

reported that those shorebirds picked the eye orbit, gills and abdominal flesh or other

tissues that contained lethal levels of PbTxs for these small shorebirds (van Deventer,

14

Atwood et al. 2012). In 2006, more than 300 sea turtles stranded along the west coast

of Florida during prolonged blooms along the coast. Some strange behavios of sea

turtles had been observed, such as swimming in circles, lack of coordination, head

bobbing, muscle twitching and jerky body movement. High densities of brevetoxins

were detected in the organs or immune cells of sea turtles, which contributed to the

stranding or death (Walsh, Leggett et al. 2010). Also, in 1996, Florida red tide caused

a significant loss in the population of the highly endangered Florida manatee (Walsh,

Leggett et al. 2010). Thus fish and sea grass can be vectors in which high

concentrations of brevetoxins accumulate. Analysis of seagrass collected in manatee’s

stomach revealed the presence of brevetoxin (Flewelling, Naar et al. 2005). In both

bloom and non-bloom periods, suspected red tide-related deaths of manatee occurred

in southwest Florida in 2002, 2003, 2005, 2007, and most recently in 2013.

Representatively, a major dolphin’s death, which occurred near the Florida panhandle

in the early 2000s, was associated with exposure to brevetoxin via food. High

concentrations of brevetoxins were detected from fish within the stomach of the

dolphin (Flewelling, Naar et al. 2005, Hoagland, Jin et al. 2009).

Over the last decade, animal studies had demonstrated the possible health effects of

both long and short terms of brevetoxin exposure. After short-term treatment, rapid

systemic distribution of brevetoxin occurred on mice after exposure to aerosolized

brevetoxin. Particularly it is concentrated in the neurologic system (Benson, Stagner

et al. 2005, Tibbetts, Baden et al. 2006). Long-term exposure of rodents (rats and

mice) to aerosols including brevetoxin showed that viral clearance in the immune

system was dysfunctional. Low level exposures (~10 pg/ml of PbTx-2 and PbTx-3) of

both asthmatic and non- asthmatic sheep resulted in decreased respiratory function.

15

However, the asthmatic sheep suffered more intense and longer lasting effects

(Abraham, Bourdelais et al. 2005, Abraham, Bourdelais et al. 2005, Abraham and

Baden 2006, Fleming, Kirkpatrick et al. 2011).

1.4.2 Biological effects of brevetoxin on human

Brevetoxin poisoning of humans can arise through food, water and air (Backer,

Fleming et al. 2003, Sikorski 2006, Fleming, Kirkpatrick et al. 2007, Grimes 2009,

Fleming, Kirkpatrick et al. 2011). NSP is associated with exposure of brevetoxin

through the consumption of contaminated shellfish. The other most common route of

exposure to the brevtoxins is inhalation (Backer, Fleming et al. 2003, Backer,

Kirkpatrick et al. 2005). The particle size of brevetoxin aerosol formed by wave

action during a red tide has a geometric mean of about 8-9 µm. In comparison,

particles that are less than 5 µm can enter the lower airway. That means only about

10-20% of these particles can enter the human lung (Pierce, Henry et al. 2003, Cheng,

McDonald et al. 2005, Cheng, Villareal et al. 2005, Cheng, Zhou et al. 2005, Pierce,

Henry et al. 2005, Fleming, Kirkpatrick et al. 2011). Nonetheless, during an active

Florida red tide season many respiratory symptoms are reported by people who spend

time on or near the west coast beaches, such as lifeguards and tourists (Cheng,

Villareal et al. 2005, Cheng, Zhou et al. 2005, Fleming, Backer et al. 2005, Fleming,

Jerez et al. 2007, Milian, Nierenberg et al. 2007). On the basis of local health care

data from Florida, a significantly higher number of hospital admission for acute and

subchronic respiratory effects such as asthma, bronchitis and pneumonia occur during

active Florida red tides period, when compared to non-active periods, particularly for

coastal residents. A comprehensive review of emergency room admissions in Florida,

16

suggested that patients require emergency care during the first hour to prevent

respiratory failure (Abraham, Plakas et al. 2008, Watkins, Reich et al. 2008). Humans

who consume contaminated shellfish can experience NSP which is characterized by

acute gastrointestinal and neurologic symptoms (Kirkpatrick, Fleming et al. 2004).

Asthma-like symptoms are related to inhalation of PbTxs by marine mammals or

people near the beach (Fleming, Backer et al. 2005, Fleming, Kirkpatrick et al. 2005).

Thus, PbTxs are responsible for a variety of negative economic, environmental, and

human health effects (Fleming, Backer et al. 2005, Errera, Bourdelais et al. 2010).

1.4.3 The effect of brevetoxin on cellular process

Multiple cellular effects are associated with brevetoxin exposure. Adverse effects

have been observed in the immune system of many species, including manatee, rat

and loggerhead sea turtle. The mechanism of brevetoxin immunotoxicity has not been

understood completely. However, several potential mechanisms have been suggested.

For example, brevetoxin inhibits the active sites of an important protease called

cathepsin (Sudarsanam, Virca et al. 1992, Kirkpatrick, Fleming et al. 2004,

Kirkpatrick, Fleming et al. 2006), which can affect the degradation of polypeptides. In

order to understand the method of the inhibition, a group developed an approach via

3D structure to search a molecule as an inhibitor for this known homologous protein

(Sudarsanam, Virca et al. 1992). They utilized the 3D structure of papain as model,

which has similar tertiary structure to cathepsins L to search for inhibitors which

filled the binding pocket using a comparative modeling technique. After searching in

the Cambridge Structural Database (CSD) of small molecules, brevetoxin B was

shown to fit in the binding pocket of papain and cathepsin L. Subsequently, both

17

papain and cathepsin L were shown to be inhibited by brevetoxin with Kis of 25 µM

and 0.6 µM, respectively.

Other effects of brevetoxin exposure include DNA damage (Murrell and Gibson

2009), reduced phagocytosis (Benson, Tischler et al. 1999), decreased plaque forming

ability (Benson, Hahn et al. 2004, Benson, Stagner et al. 2005) and chromosomal

aberration (Sayer, Hu et al. 2006). Several studies have indicated that oxidative stress

may occur as the cellular response to brevetoxin exposure. For instance, After the

human U-937 cells were treated with PbTx-2, the amount of glutathione was

decreased while oxidative stress occurred (Walsh, Leggett et al. 2009). Other studies

on marine organisms revealed a link between brevetoxin exposure and oxidative

stress. The activity of the oxidative stress marker superoxide dismutase (SOD) was

detected and correlated to the plasma brevetoxin levels and reactive oxygen in

rescued Florida manatees after exposure to a red tide bloom (Walsh, Stuckey et al.

2007, Walsh, Butawan et al. 2015). When compared to healthy turtles, a two-fold

increase in SOD was observed in rescued loggerhead turtles which correlated with

plasma brevetoxin levels at 500 ng / ml (Walsh, Leggett et al. 2010). Brevetoxin

exposure at 5.4-15.0 µg/L increased catalase activity in coral larvae and lipid

peroxidation in coral larvae and fish gills (Woo, Liu et al. 2006, Ross, Ritson-

Williams et al. 2010). Furthermore, PbTx-2 was shown to induce apoptosis in human

Jurkat cells (Walsh, Leggett et al. 2008). When Jurkat cells exposed to 5-10 µg/ml

PbTx-2 or PbTx-6, the decrease of cellular metabolic activity was observed. After 24h,

the viability of cells that were treated with PbTx-2 decreased. In contrast, apoptosis

was increased under PbTx-2 or PbTx-6 exposure. Unfortunately, the mechanism for

brevetoxin induced oxidative stress is still unknown.

18

1.5 LSP compounds bind to transmembrane protein

The best-known biological effect of brevetoxins is the activation of voltage-gated

sodium channel (VGSC), a transmembrane protein, which induces the ion influx

resulting in numerous physiological effects.

The VGSC is composed in large part of transmembrane α-helices, and membrane-

integral α-helix peptides are considered as common interacting motifs of LSPs.

Voltage-gated sodium channel is essential for the initiation and propagation of action

potentials in excitable cells. Voltage-gated sodium channel has long been believed to

be the principle mammalian target of the brevetoxins. In addition, because of the

similarity of yessotoxin to brevetoxin, it has been suggested that yessotoxin increased

the permeability of sodium ions in cell membrane, resulting in the symptoms of CFP.

For example, the α-helices of glycophorin A (GpA), a membrane protein found in

erythrocytes, have been used as a model system for evaluating the interaction between

LSPs and α-helices. The association between α-helical glycine and valine residues of

GpA result in dimer formation in membrane environments (Bormann, Knowles et al.

1989). The association of LSPs such as YTX, desulfated YTX, PbTxs and artificial

polyethers with GpA results in disruption of the oligomers or dimers. The disruption

can be evaluated by SDS–PAGE. The results demonstrated that the LSPs interact with

the transmembrane portion of glycophorin A to induce the dissociation of oligomers

of the protein. In order to quantify the interaction of LSPs with GpA, surface plasmon

resonance (SPR) and saturation transfer difference NMR were applied. The

dissociation constants (Kd) of YTX and desulfated YTX with a transmembrane

domain peptide of GpA were determined to be in the submillimolar range (Ujihara,

19

Oishi et al. 2008). All results suggested that hydrophobic matching is important for

LSP binding to transmembrane α-helices. Moreover, taken together, it indicated that

hydrophobic interactions play a key role in molecular recognition of the α-helical

peptides by LSPs.

Both brevetoxins and ciguatoxin bind to and activate the VGSC at site 5. These site 5

toxins induce channel opening at normal resting potentials leading to a change in the

biophysical properties of the VGSCs. Like brevetoxins, VGSC opening at normal cell

resting membrane potentials results in an influx of Na+ ions and cell depolarization.

Although ciguatoxin acts at the same receptor site of the Na+ channel as brevetoxin,

the affinity of brevetoxin for sodium channel is around 30 times lower than that of

CTX-1. In addition, CTX-1 competitively inhibits the binding of brevetoxin to the

voltage-dependent Na+ channel of rat membranes. As a consequence, ciguatoxin

cause the an abnormally prolonged Na+ channel opening in nerve membranes,

slowing the nerve conduction velocity (Lehane and Lewis 2000, Kumar-Roiné,

Matsui et al. 2011). In addition, compared to the short-term effects on nerve and

muscle tissue by brevetoxin, ciguatoxin induces long-term nerve damage. The plasma

membrane is unable to maintain the internal environment of cells and volume control,

resulting in cell and mitochondrial swelling and bleb formation. Thus, ciguatoxin is

responsible for the disruption of important ion exchange system (Lehane and Lewis

2000). Fortunately, intravenous (IV) mannitol infusion is the most common treatment

for CFP (Friedman, Fleming et al. 2008). Mannitol is given at a dose of 0.5 to 1.0

g/kg body weight over 30-45 minutes. However, it must be administered within 24

hours of exposure to be effective. Also, the osmotic reduction of neuronal edema was

20

indicated to mediate the effect of mannitol infusion (Pearn 2001, Friedman, Fleming

et al. 2008).

Last but not least, maitotoxin, a water soluble toxin, acts on calcium channels

(Taglialatela, Amoroso et al. 1986). Treatment of cells with low concentrations of

maitotoxin results in activation of voltage sensitive calcium channels demonstrating

that maitotoxin affects the uptake of calcium ion in cells. However, no affinity was

found by maitotoxin for voltage-gated sodium channel through binding experiment on

rat brain cortical synaptosomes. Furthermore, it is also reported maitotoxin is

associated with neurotransmitter, hormone release, phospholipid metabolism and

smooth muscle contractions (Schettini, Koike et al. 1984, Berta, Phaneuf et al. 1988,

Gusovsky and Daly 1990).

1.6 The interaction between PbTxs VGSC at the molecular level

1.6.1 PbTxs exert their effects through specific binding of PbTx to the VGSC

Previous work has shown that there are multiple distinct receptor sites for neurotoxins

in sodium channels. For example, receptor site 1 binds the inhibitors tetrodotoxin and

saxitoxin, which block ion transport through the sodium channel. Grayanotoxin and

the alkaloids veratridine, batrachotoxin and aconitine bind to receptor site 2, which

cause repetitive firing and persistent activation of sodium channels (Catterall 1980,

Catterall, Morrow et al. 1981, Catterall 1986). Receptor site 3 binds the β-scorpion

toxins and sea anemone toxins, which can inhibit sodium channel inactivation and

enhance persistent activation of sodium channels by veratridine and other toxins

acting at receptor site 2 (Tejedor and Catterall 1988, Rogers, Qu et al. 1996).

Receptor site 4 binds ß-scorpion toxins from American scorpions, which can shift the

21

voltage dependence of sodium channel activation (Catterall 1977). These other

neurotoxins that bind to the sodium-voltage gate channel failed to displace 3H labeled

PbTx from its binding site. The previous experiments led to the conclusion that the

PbTxs bind to a unique site in the VGSC that was termed site 5 (Poli, Mende et al.

1986). The specific binding is reversible and temperature-dependent. Through

analysis, PbTx-3 binding was determined have a Kd of 2.9 nM (at 4°C). Equilibrium

binding was also observed after incubation of 3H labeled PbTx-3 with Tilapia brain

synaptosomes with a Kd of 6.1 nM (Stuart and Baden 1988). It was also determined

that PbTx-3 can be displaced by PbTx-1 and PbTx-2 at specific binding site.

Electrophysiological studies revealed that specific binding of PbTx to the VGSC

results in membrane depolarization in a dose dependent manner (Huang, Wu et al.

1984). When PbTx is incubated with crayfish giant axons, the extent of maximum

depolarization is about 40 mV, with an EC50 of 1.7 nM (Huang, Wu et al. 1984). Thus,

PbTxs cause depolarization of excitable membranes, facilitating the inward flow of

sodium ions into the cells (Trainer and Baden 1991, Dechraoui, Naar et al. 1999).

Binding of PbTxs to the sodium channel causes a shift the channel activation potential

to more negative values, inhibits channel inactivation and prolongs mean open times

of the channel (Jeglitsch, Rein et al. 1998). Thus, PbTxs disrupt the normal

transmission of electrical signals within the neurological system. A massive release of

neurotransmitter is stimulated by the depolarization of nerve terminals, which results

in a wide range of responses in the organs. Therefore the consumption of seafood

contaminated with PbTxs has the potential to disrupt normal neurological systems

causing the illness clinically described as NSP (Twiner, Bottein Dechraoui et al.

2007).

22

1.6.2 Specific interactions between brevetoxin and the VGSC

The VGSC is composed of an α-subunit and two β-subunits. The α-subunit is the

major subunit and made up of four domains. The four domains of the channel

containing six transmembrane α-helices with the connecting amino acid sequence

between helices 5 and 6 coming together to line the pore through which the ions travel

(Heinemann, Terlau et al. 1992, Hille 2001). Early studies, which helped to define the

interaction between the PbTxs and the VGSC at the molecular level included structure

activity studies, conformational analysis and photoaffinity labeling of the receptor

(Poli, Mende et al. 1986, Trainer and Baden 1991, Trainer, Thomsen et al. 1991, Rein,

Baden et al. 1994).

Brevetoxin-A and brevetoxin-B bind to the same unique site of the VGSC at site 5

which is located on domain IV of the α-subunit of the channel. Using photoaffinity

labeled PbTx in rat brain synaptosomes the PbTx high affinity receptor site was

localized to the α-subunit. After binding of the photoaffinity probe, a p-azidobenzoate

tritium-labeled brevetoxin derivative, to the VGSC and photoloysis, it was shown that

the VGSC was covalently labeled with the PbTx-3 derivative. After digestion with

proteolytic enzymes, antibody mapping of the proteolytic fragments localized the

covalent incorporation of this neurotoxin to the sodium channel α-subunit on the

extracellular side of S5 in domain IV and on the extracellular end of the S6 in domain

I (Rein, Baden et al. 1994, Trainer, Baden et al. 1994).

Neuronal tissues in marine mammal have similar sodium channel density as terrestrial

mammals. Brevetoxin (PbTx-3) binds to manatee brain synaptosomes with nanomolar

23

affinity at 37 oC. (Trainer and Baden 1999). The dissociation constant (Kd) is 7.5±2.5

nM for manatee synaptosomes, which is a similar affinity to terrestrial mammals

(Trainer and Baden 1999). Since 1982 to 2005, hundreds of manatees were found

dead or dying on the beaches of the Florida west coast when high densities of K.

brevis were present, including 39 deaths in 1982, 149 deaths in 1996 and 34 deaths in

2002. Unbelievably, in 2005 it is reported that 85 manatees died because of K. brevis

(O'Shea, Rathbun et al. 1991, Bossart, Baden et al. 1998). In Assuming 11 pg

brevetoxin was produced from a single cell (Stuart and Baden 1988). If the affinity of

manatee for brevetoxin is 7.5 nM, approximately, 3 x 105 cell/L brevetoxins are

required. Whereas in fact concentration of brevetoxin cells at that period persisted at

100,000 cells/L. This discovery indicated that manatees were exposed to a lethal dose

of brevetoxin in 1996 (Trainer and Baden 1999). Neuronal cells, skeletal muscle cells

and cardiac cells are all excitable tissues, which contain VGSC to mediate the rapidly

activation and inactivation of Na+ influx. A comparative study examined the binding

properties of brevetoxins with skeletal muscle and cardiac sodium channel α-subunit

isoforms of human embryonic cells. Brevetoxin B was found to have isoform

selectivity with cardiac channel and skeletal muscle channel (Dechraoui and Ramsdell

2003). It is determined that the inhibitory constants Ki for PbTx-3 were 4.7 nM for

skeletal muscle channels, while it is 20.5 nM for cardiac sodium channels.

1.7 The effect of brevetoxin on economy

As a consequence of a bloom along coastal waters, local economies suffer not only

direct losses to fishing and tourism but also economic effects associated with animal

death, clean-up, rescue and rehabilitation, and public health also cause additional

24

losses. A single toxic bloom can result in as much as $22 million in lost revenue from

tourism and related business (Dyson and Huppert 2010, Anderson, Moore et al. 2015).

In 2000, a large bloom of K. brevis occurred in Galveston County, Texas. It required

$18 million for direct cleanup and subsequent losses to fishing and tourism (Evans

and Jones 2001). The economic costs caused by the harmful effect of K. brevis are

estimated to exceed $38 million annually (Twiner, Bottein Dechraoui et al. 2007). A

cost model using the data on emergency room visits for respiratory damage, the K.

brevis density, measurement of pollutant, annual population change and tourism

estimated that the cost of illness associated with K. brevis in Sarasota county alone

can range from $0.5-$4 million (Hoagland, Anderson et al. 2002, Hoagland, Jin et al.

2009, Kirkpatrick, Bean et al. 2010). Furthermore, during an active Florida red tide

period the costs resulting from lifeguard absenteeism can reach up to $100,000 in

Sarasota Beach alone. In 2014, Hoagland estimated that annual costs of illness caused

by Florida red tide ranged from $60,000 and $700,000 per year. However, it is

assumed that this cost could be increased to $ 1 million per year by several long-last

Florida red tide. Furthermore, in future the cost of illness would range between $2 and

$24 million under a worst case scenario (Hoagland, Jin et al. 2014).

1.8 Brevetoxins play an important role in K. brevis, but it is still elusive

Because PbTxs are retained in K. brevis cells, it has been presumed that they play a

functional role for K. brevis. Although a primary cellular function for PbTxs has

remained elusive, it is reported that the responses to environmental conditions, such

as salinity, influence the growth potential and toxin production of harmful

dinoflagellates (Grzebyk, Béchemin et al. 2003, Magaña, Contreras et al. 2003). In

2011, it was proposed that PbTxs play an important role in osmoregulation by K.

25

brevis. It was found that osmotic stress triggered PbTx production by K. brevis in

three K. brevis clones under variable salinity conditions that result in changing

osmotic stress. A rapid decrease in salinity from typical oceanic conditions of 35 ppt

to approximate coastal conditions of 27 ppt resulted the concentration of brevetoxin

per cell increasing within 5 days under hypoosmotic stress and provided evidence to

suggest PbTxs play a role in osmoregulation. Furthermore, in order to understand the

response of K. brevis to osmotic stress, a time course experiment to measure changes

in cell volume and PbTxs production was performed. <3h, 24h, 6d, 67d, and 111d

after the rapid salinity shift PbTxs content was examined. Brevetoxin increased within

3 h after the hypoosmotic stress and continued to increase through day 6. After 6 days

the concentration of PbTxs reached the highest level (Errera and Campbell 2011).

However, in 2012 this conclusion was challenge by several other research groups who

attempted to repeat Errera and Campbell’s experiments. Two separate laboratories

reported that a sudden decrease in salinity from 35 ppt to 27 ppt, did not trigger

brevetoxin production. These results were consistent across each of eight K. brevis

strains (Sunda, Burleson et al. 2013). Consequently, this debate generated much

attention from researchers. Still, the cellular function of PbTxs is under debate.

Chemical and predatory defense has been proposed as the reasons for brevetoxin

production by K. brevis. Feeding experiment experiments using PbTxs have been

conducted to better understand how K. brevis escapes predation pressure. Rotifers are

common microzooplankton grazers which co-exist in coastal water with K. brevis.

Compared to a known palatable food for rotifers, when K. brevis cells were offered as

a sole diet for rotifers, all rotifers stopped grazing and breeding. However, it is not

clear that the deterrence of grazing was a result of the presence of PbTxs. Rotifers fed

26

with yeast containing pure PbTx-2, PbTx-3 and PbTx-9 were not deterred from

grazing (Kubanek, Snell et al. 2007). Thus, defending K. brevis against grazing by

may or may not be a role for brevetoxins.

1.9 The significance of studying the dinoflagellates

In the past decades, scientists have made significant progress in determining the

effects and the underlying mechanisms of dinoflagellate toxins on higher organisms.

Because of the extensive damage and pharmacological effects caused by K. brevis,

there is considerable interest in understanding the function of PbTxs. If a metabolic or

biochemical role for brevetoxins can be identified, this may provide for more accurate

prediction of bloom dynamics and toxicity. The role of brevetoxin in cells can lead to

more accurate predictions, mitigation and management of toxic blooms. While

numerous hypotheses have been proposed and several studies performed, the intrinsic

role of dinoflagellate toxins remains unknown In addition; the discovery of the native

role of brevetoxin in K. brevis may provide insight into understanding the role of

other polyether compounds in dinoflagellates such as ciguatoxin, maitotoxin and

yessotoxin.

1.10 Research objectives

The research will focus on identifying the intrinsic biochemical function of PbTxs and

identification of a molecular receptor for brevetoxin in K. brevis. Localization of

PbTxs to a subcellular organelle and identification of a target protein from K. brevis,

which interacts with PbTxs as well as comparison of biochemical and metabolic

indicators in K. brevis strains with vastly different toxin content, may point towards a

native function for PbTxs.

27

Objectives

1. To localize brevetoxin in K. brevis using a fluorescently labeled brevetoxin probe.

2. To identify one or more target protein(s) of brevetoxins from K. brevis.

3. To characterize interaction between brevetoxin and its native receptor protein

4. Compare and contrast various metabolic and biochemical parameters in two strains of

K. brevis which have a tenfold difference in toxin content.

28

Chapter 2 Localization of PbTxs to a subcellular organelle and identification of a

target protein from K. brevis

2.1 Localize PbTxs to one or more organelles or cellular structures in K. brevis

2.1.1 Objective

The localization of brevetoxin to one or more organelles in K. brevis cells should

provide clues as to the intrinsic role of brevetoxin in K. brevis. In this section, we

describe the use of a fluorescently labeled brevetoxin probe along with fluorescence

microscopy to identify the site of localization of brevetoxin.

2.1.2 Introduction

Labeling of biological molecules is one of the most common imaging methods used in

bioanalytical fields, such as medicine, pharmacy, cellular biology and environmental

science. Labels can be radioactive or molecules with absorption or fluorescence.

Fluorescence techniques have been used for biological imaging for many decades.

Thousands of fluorescence probes are used to stain the organelles in living cells and

many fluorescence stains are commercially available. For instances, DAPI (4’, 6-

diamidino-2-phenylin-dole) is a fluorescent dye which is specific for DNA. In the

1970s DAPI was discovered to stain the DNA of yeast cells. It was demonstrated that

DAPI fluorescence was sensitive to DNase (Williamson and Fennell 1975). DAPI

forms a fluorescent complex through its high affinity binding of DAPI with the minor

grove of A-T rich sequence of DNA (Williamson and Fennell 1975). Thus, DAPI can

be a biological stain for nucleic acids and chromosomes (Lin and Alfi 1976,

Schweizer 1976, Schweizer 1976, Lin, Comings et al. 1977). Blue-fluorescent

29

Hoechst 33342 dye also stains the nuclei and hematoxylin is a stain for nuclei in

living cells (Fischer, Jacobson et al. 2008). Rhodamine is one of numerous

histological stains, which have been utilized for many years as a specific probe to

localize the mitochondria in living cells. Rhodamine 123 also can be used in detection

of the energy state of isolated mitochondria (Azzone 1972, Johnson, Walsh et al.

1980). MitoFluor™ Far Red 680 accumulates in mitochondria, and its fluorescence

(emission maximum 700 nm) was red. Green-fluorescent LysoTracker® Green dye

accumulates in the lysosomes.

The dyes described above are general dyes for staining organelles. Reactive reporter

dyes may be used to detect specific classes of molecules within a cell. In 2008, a type

of fluorescent probe called MitoPY1 for imaging hydrogen peroxides within

mitochondria of live cells was determined. MitoPY1 was synthesized with a peroxide-

responsive element and mitochondrial targeting phosphonium moiety to localize

hydrogen peroxide to cellular mitochondrial (Dickinson and Chang 2008). A

rhodamine modified as an electron donor (4-amino- or 4- hydroxyphenyl ether), is

used for detection of highly reactive oxygen species (ROS) in mitochondria of live

cells. thereby ROS should cause cleavage of the ether moiety resulting in fluorescence

in mitochondria (Koide, Urano et al. 2007). Thus, these indirect and modified

fluorescent probes are utilized to selectively detect biomolecules in live cells.

The fluorescent tagging of specific molecules is a promising technique for localizing

molecules in live cells. For example, immunofluorescent staining is able to detect

biomolecules in living cells as long as the specific antibody is provided. The

fluorochrome-labeled antibody is used to stain intracellular biomolecules as a

30

common and direct method. Permeabilization of the plasma membrane is an essential

and often problematic, step in this method (Suzuki, Matsuzaki et al. 2007). For

additional examples, labeled oligonucleotide probes were used to image individual

mRNA molecules in fixed cells, which made each mRNA molecule visible as

fluorescent microscopy (Raj, Van Den Bogaard et al. 2008). Using a designed and

synthesized fluorescent probe included a small receptor domain and ligand label some

recombinant proteins which contains four cysteines at 5 positions of an α helix. The

small receptor domain is composed of six natural amino acids that could be

genetically mutated-bind to proteins of interest. The ligand is membrane-permeant

and nonfluorescent until it binds specifically with high affinity to the tetracysteine

domain (Griffin, Adams et al. 1998).

In order to understand the biochemical function for brevetoxins, our first goal is to

localize brevetoxins to one or more organelles or cellular structures in K. brevis.

Consequently, using a fluorescently tagged brevetoxin probe is an effective and direct

approach to localize PbTxs in K. brevis. The site of localization can be visualized via

fluorescence microscopy

The synthesis of a fluorescently labeled brevetoxin probe has been reported

previously (Cassell, Chen et al. 2015). Briefly, Alexa Fluor® 488 alkyne (4) is used as

fluorescent dye linked to brevetoxins, which are highly lipophilic molecules (Figure7).

The fluorescently labeled PbTx-2 probe (5) was prepared by coupling brevetoxin with

an Alexafluor alkyne derivative (4). A thiol-Michael reaction between PbTx-2 (1) and

an azidothiol (2) provided a azide functionalized thiazepine linker (3), which was

subjected to a 1, 3-dipolar cycloaddition reaction with Alexa Fluor 488 alkyne (4).

31

The Alexa Fluor dye was chosen specifically because it is cell-impermeable. Thus, if

the brevetoxin - Alexa Fluor® 488 Alkyne conjugate can penetrate the membranes, it

is only because of brevetoxin. The thiol-Michael reaction has been described as a

“click” reaction (Nair, Podgorski et al. 2013). “Click chemistry” is commonly used to

combine an azide labeled molecule and an alkyne labeled molecule. A “click

“reaction is a fast reaction which can be performed under mild conditions (Nair,

Podgórski et al. 2013). Subsequently, the probe was incubated with two K. brevis

strains, a high toxin producing strain and a low toxin producing strain. Stained cells

emitting light at 500-540 nm were observed using fluorescence microscopy. Thus

using live cell imaging, the location of brevetoxins in K. brevis cells can be identified.

Figure 7 Synthesis of fluorescent brevetoxin probe. For brevity, only the K-ring of PbTx-2 is shown. a) MeOH, b) tBuOH/H2O/DMF, CuSO4·-5H2O, sodium ascorbate

2.1.3 Results and discussion

In order to determine the location of brevetoxin in K. brevis, the fluorescent probe (5)

and Alexa Fluor® 488 Alkyne (4) were each incubated for 1 hr with K. brevis living

cells. After washing the cells with culture medium, the cells were visualized by using

32

a fluorescent microscope equipped with a 450–490 nm band pass/515 nm long pass

EX/EM filter set. Chlorophyll auto fluorescence (red) could be clearly seen in both

treatments (Figure 8). However, incubation with the fluorescent PbTx-2 probe also

demonstrated localization of the fluorescent PbTx conjugate (yellow) to the

chloroplasts (Figure 8 A and B). Additive color is produced by combining a number

of primary colors such as green, red and blue in equal proportions. Thus, although

Alexa Fluor® 488 alkyne is a green dye, based on the additive color mixing, the green

color combines with red color resulting in yellow. However, the control experiment,

with Alexa Fluor® 488 alkyne alone, failed to label K. brevis cells (Fig 8 C). This

experiment demonstrated that PbTxs localize to the chloroplast of K. brevis

Figure 8 The photograph of localization of brevetoxin in K. brevis live cells (A) Fluorescent brevetoxin derivative (0.4 mg / ml) was incubated with live K. brevis culture for 30 mins in dark. The cells were observed through 40X lenses of a fluorescent microscope equipped with a 450–490 nm band pass/515 nm long pass EX/EM filter set. (B): The zoom in image of labeled cells. (C): The zoom in image of cells in control experiment: Alexa Fluor® 488 Alkyne (0.06 mg / ml) was incubated with live K. brevis culture for 30 mins in dark

The finding is consistent with the localization of okadaic acid (OA) (Fig 9), a marine

toxin produced by some dinoflagellates of the genera Prorocentrum and Dinophysis

(Zhou and Fritz 1994). The location of OA has been identified first with

immunofluorescence. Toxin-producer cells were incubated with anti-OA fluorescein

isothiocyanate (FITC) - conjugated antibody. Fluorescence was observed at the

33

plastids. In order to obtain even more specific localization, immunogold labelling of

anti-OA antibody was used. Using the immunofluorescence micrographs through

transmission electron microscope operating at 80 kV, the gold particles were visible

with the pyrenoid region of the chloroplast. Thus, it had been proposed that OA was

localized primarily within chloroplasts of toxin-producing Prorocentrum species

(Zhou and Fritz 1994).

Figure 9 The structure of okadaic acid

2.2 Isolation of a target protein from K. brevis as PbTxs natural receptor

2.2.1 Objective

Identification of a brevetoxin binding protein in K. brevis may provide some clues to

the intracellular function of PbTxs in K. brevis cells.

2.2.2 Introduction

Photoaffinity labeling (PAL) provides a direct and general method to identify binding

proteins. Photoaffinity labeling can directly identify the interacting site at the

molecular level. In photoaffinity labeling, a new covalent linkage is created between a

light-sensitive, detectable ligand and a biopolymer upon irradiation, in a reversibly

bound state (Dormán and Prestwich 2000). A typical photoaffinity probe incorporates

three groups, a ligand, a photoreactive group, and a tag for purification. The

photoreactive groups should have some essential features: the photoreactive groups (1)

must be stable under ambient light with an activation wavelength longer than 300 nm

34

(Dormán and Prestwich 2000). (2) should preferably provide a single covalent adduct

between the probe and target protein avoiding release of an activated form that results

in non-specific labeling (3) have an activated form that reacts with C-H groups and X-

H bonds. The most reliable and common high efficiency labeling of target proteins

are tetrafluorophenyl azides (AZ), trifluoromethylphenyl diazirines (DIA) and

benzophenone (BP) (Fleming 1995, Weber and Beck-Sickinger 1997)(Figure10 ).

These photoactiveatable groups have been used extensively to identify the target of

drugs, to determine the affinity and selectivity of the drug-target interaction, and to

identify the binding site on the target.

Figure 10 The most reliable and common high efficiency labeling of target proteins in photoaffinity probe

Although less common, photoaffinity labeling has also been used in non-

pharmaceutical applications as well, with several studies using DIA as the photophore.

In 2003, using DIA modified photoaffinity probe that attached to 2’ -position of the

sugar moiety, a potential binding site to the receptor molecule, successfully detected

the potential membrane receptor proteins for leaf movement of legumes (Sugimoto,

Fujii et al. 2004). DIA that linked to maitotoxinin with a biotin detectable group was

used to identify a binding protein for maitotoxin in red blood cells (Konoki,

Hashimoto et al. 2009). Although the target protein has not been characterized, based

on the SDS-PAGE, a 23 kD protein was specifically labeled. Competition binding

experiments with PbTx-2 demonstrated that both toxins bound to the same site on this

35

protein. Thus, the use of a PbTx-2 photoaffinity probe could be a direct and effective

method to discover the target protein of PbTxs in K. brevis.

The synthesis of the photoaffinity probe has been previously reported (Cassell, Chen

et al. 2015). The photoaffinity probe (10) was consisted of a photolabile diazirine and

a biotin tag. In order to isolate a target protein, the biotin-streptavidin interaction was

used to separate the target protein from others. Briefly, the known aldehyde (6)

(Hashimoto, Hatanaka et al. 2001) was alkylated with 1-azido-6-bromohexane to

provide azide (7). “Click chemistry” was used to link the azide functionalized

diazirine with an alkyne functionalized biotin to provide (8). Reductive alkylation of

the aldehyde moiety with cystamine, followed by reduction of the intermediate

disulfide, provided the thiol (9). Thiol (9) then underwent a thiol-Michael reaction

with PbTx-2 (1), followed by cyclization to the thiazapeine provided photoaffinity

probe (10) (Figure 11).

36

Figure 11 The synthesis of brevetoxin photoaffinity probe (10). For brevity, only the K-ring of PbTx-2 is shown. a) K2CO3, KI, DMF; b) tBuOH/H2O/DMF, CuSO4·5H2O, sodium ascorbate; c) (NH2CH2CH2S)2, NaBH4, EtOH; d) DTT, MeOH; e) MeOH; 2.2.3 Results and Discussions

A homogenate of K. brevis cells was separated into soluble and membrane fractions

by centrifugation. These fractions were each incubated either with the photoaffinity

probe (10) or the photoaffinity label (8), followed by photolysis and protein isolation

using streptavidin immobilized on magnetic beads. After incubation of the brevetoxin

photoaffinity probe (10) with the soluble protein fraction and photolysis no proteins

were observed in SDS PAGE after purification on streptavidin beads. However,

incubation and photolysis of the membrane fraction with the brevetoxin photoaffinity

probe (10) consistently revealed a protein with an apparent mass of 27 kD when

analyzed by SDS-PAGE (12). A control experiment with photoaffinity label (8) alone

did not yield any bands by SDS-PAGE.

37

The 27 kD bands were subjected to digestion with trypsin. After analysis of the

excised band by ESI LC-MS/MS followed by BLAST (basic local alignment search

tools) analysis of the amino acid sequence against NCBI non-redundant database, it

was determined that there were two thylakoid proteins which interacted with

brevetoxin: the light harvesting complex II (LHC II) of the photosynthetic apparatus

and thioredoxin (Trx). The identified peptide fragments and their closest match in the

BLAST analysis are shown in Table 2. Subsequently, we determined that our

photoaffinity probe also binds to the LHC II of spinach (Figure13 lane 6).

Figure 12 SDS-PAGE of protein fractions from photoaffinity experiments. K. brevis membrane fractions incubated with photoaffinity probe (10) or (8). Lane 1: molecular weight markers. Lane 2: K. brevis membrane fraction incubated with (10) (7 mg protein). Lane 3: pre-incubation membrane fraction (7 mg protein). Lane 4: K. brevis homogenate incubated with (8) (18 mg protein). Lane 5: pre-incubation membrane fraction (6 mg protein). Lane 6: spinach homogenate incubated with (10) (16 mg protein)

Table 2 The peptide sequence of 27 kD band and the proteins with matching sequences.

Protein name Organism Peptide sequence

LHCII Emiliania huxlei GGKPGYFPTFK LHCII Emiliania huxlei PGYFPTFK LHCII Emiliania huxlei LAMLGLFSLISEAK LHCII Emiliania huxlei VPGAVPALAGK

Thioredoxin Emiliania huxlei IIHLTDDSFDTDVLK Thioredoxin Emiliania huxlei MIAPILDEIADEYQGK Thioredoxin Emiliania huxlei GIPTLLLFK

Thioredoxin 1 Aliivibrio salmonicida LNIDQNAGTPPK

38

Thylakoids are membrane-bound structures that are located in chloroplast stroma. The

light-dependent reactions of photosynthesis occur in the thylakoids. The thylakoid is

composed of a thylakoid membrane surrounding a thylakoid lumen. In the thylakoid

membrane the photosynthetic pigments are bound to proteins which are embedded in

the membrane. There are many integral membrane proteins that play important roles

in light harvesting and the light dependent reactions of photosynthesis take place in

thylakoid membranes. Photosystems I and II, cytochrome b6f complex and ATP

synthase are four major protein complexes in the thylakoid membrane. LHC is a

complex of subunit protein that is a part of a larger supercomplex of a photosystem.

LHC II surrounds the photosynthetic reaction center in thylakoid membranes to both

harvest and transfer light energy to photosystem II and to protect it from oxidative

damage that is caused by excess sunlight. The molecular weight of LHC II is 27 kDa.

It is highly conserved across species. It is essential for both light harvesting and non-

photochemical quenching (NPQ). NPQ is a mechanism to quench chlorophyll

fluorescence and dissipate the excess chemical energy as heat rather than chlorophyll

fluorescence which results in the formation of ROS. Thus, an important function of

LHCII is to prevent oxidative damage to photosystem II. LHCII binds to 14

chlorophyll and 4 carotenoid molecules. These pigments are used for light harvesting

and energy dissipation. In terrestrial plants, the four carotenoids are two luteins,

neoxanthin and zeaxanthin or violaxanthin. In dinoflagellates the zeaxanthin and

violaxanthin are replaced by dinoxanthin (D) and diadinoxanthin (DD) respectively.

Lutein and neoxanthin are replaced by fucoxanthin in K. brevis (Demers, Roy et al. ,

Arsalane, Rousseau et al. 1994, Moisan, Olaizola et al. 1998, Vijayan and Somayajula

2014). The zeaxanthin/violaxanthin interconversion facilitates light energy

39

dissipation through the xanthophyll cycle (Figure 13). Under high light conditions, the

violaxanthin is converted to zeaxanthin in a process catalyzed by the enzyme

violaxanthin de-epoxidase (VDE). It has been assumed that zeaxanthin binds to LHC

II which undergoes a conformational change moving zeaxanthin into close proximity

to the chlorophyll molecule permitting the quenching of chlorophyll fluorescence.

Under low-light conditions zeaxanthin is converted back to violaxanthin by the

enzyme zeaxanthin epoxidase (ZE) (Fuciman, Enriquez et al. 2012). However, it is

not clear if violaxanthin is released from LHC II or converted within LHC II.

Figure 13 Xanthophyll cycle

Under excess light condition, photosynthesis begins; the lumenal side of the thylakoid

membrane experiences a significant drop in pH (from 8 to 5). NPQ is initiated by this

drop in pH on the lumenal side of the thylakoid membranes in response to light. In

oreder to determine if brevetoxin binding to LHCII is pH dependent, the binding of

brevetoxin photoaffinity probe to LHCII in spinach membranes was examined by

using a pH range from pH 2 (simulating high light conditions) to pH 8 (simulating

low light conditions). After incubation of the crude spinach homogenate with the

brevetoxin photoaffinity probe, photolysis at 312 nm for 20 min and affinity

40

purification on magnetic streptavidin beads, SDS PAGE analysis was performed on

the eluant. Inspection of the SDS PAGE gel revealed a pH dependence on binding

(Figure14). In the Figure 14, the red box is actin added as an internal standard for

normalization. The green box is the 27 kD band contained LHCII. The orange box is

the smaller band which has numerous photosystem I and II membrane proteins,

Rubisco small subunit, thioredoxin and a serine/threonine protein kinase. Brevetoxin

bind more strongly to 27 kD protein at low pH (or conditions which simulate high

light). Nevertheless, at high pH (or conditions which simulate low light) brevetoxin

binds with several lower molecular weight proteins (below 20 kDa, Figure 14) The

lower molecular weight band was also observed in every experiment. In order to

determine the relative intensities of each band, a constant amount of actin was mixed

with the eluant as an internal standard (Table 3). The lower molecular weight band

was also subjected to digestion with trypsin followed by MS/MS analysis. After

analysis of the digested band by ESI LC-MS/MS followed by BLAST analysis of the

amino acid sequence against NCBI non-redundant database, five different

photosystem I and six different photosystem II membrane proteins were identified., as

well as three proteins which are located in the stroma: thioredoxin, a protein kinase,

and Rubisco (Table 4). Thus, it appears that at high pH or high light, brevetoxin may

be simply released from LHC II into the thylakoid membrane where it might interact

with many proteins.

41

Figure 14 Spinach homogenate was incubated with the photoaffinity probe in different pH buffer (Lane 2: pH=8, Lane 3: pH=6, Lane 4: pH=4, Lane 5: pH=2). Red Box: actin (added as an internal standard) for normalization; Green box: 27KD LHCII; Orange box: numerous photosystem I and II membrane proteins, Rubisco small subunit, thioredoxin and a serine/threonine protein kinase

Table 3 The intensity of all bands on SDS-PAGE gel

Intensity of Actin

Intensity of 27 kD

band

Intensity of 12 kD

band

The ratio of 27 kD band to 12 kD band

The ratio of 27 kD band to Actin

The ratio of 12 kD band to Actin

The ratio of

two bands to

actin pH=2 26641 19770 10376 1.91 0.74 0.39 1.13

pH=4 31309 22237 13294 1.67 0.71 0.42 1.13

pH=6 28672 18287 13010 1.40 0.64 0.45 1.09

pH=8 26675 9922 13092 0.76 0.37 0.49 0.86

Table 4 The peptide sequence of the 12 kDa band the proteins with matching sequences

Protein name Organism Peptide sequence

Thioredoxin Escherichia coli LNIDQNPGTAPK Thioredoxin Escherichia coli GIPTLLLFK

Protein kinase Burkholderia cepacia GG4 VATVELPR

Rubisco Spinacia oleracea YETLSYLPPLTTDQLAR

Rubisco Spinacia oleracea QVDYLLNNK Rubisco Spinacia oleracea IIGFDSNR

Photosystem I subunit F Spinacia oleracea DIAGLTPCK

Photosystem I subunit F Spinacia oleracea LYADDSAPALAIK Photosystem I subunit F Spinacia oleracea SYLIAIRDEK

Photosystem I subunit L Spinacia oleracea TAVNPLLR

Photosystem I subunit H Pelargonium x hortorum FFETFAAPFTK Photosystem I subunit K Oryza sativa Japonica Group FGLAPSANR

Photosystem I subunit E

Phillyrea latifolia

GVGSVVAVDQDPK

42

Photosystem II protein D2 Acutodesmus obliquus AYDFVSQEIR

Photosystem II protein D2 Acutodesmus obliquus AAEDPEFETFYTK

Photosystem I subunit S Spinacia oleracea ENELFVGR Photosystem I subunit S Spinacia oleracea FVDEPTTGLEK

Photosystem I subunit Q Spinacia oleracea VSASEILNVK

Photosystem I subunit R Agave tequilana GVYQFVDK Photosystem II protein V Spinacia oleracea SFADIITSIR

Photosystem II protein V Spinacia oleracea PNEYFTESR

Photosystem II protein V Spinacia oleracea QGIPLITGR Photosystem II protein V Spinacia oleracea FDSLEQLDEFSR

Cytochrome f Spinacia oleracea EVVDIIPR

LHC II Oryza sativa Japonica Group VGGGPLGEGLDK

LHC II Spinacia oleracea VAGGPLGEVIDPLYPGGSFDPLGLADDPEAFAELK

Many of the proteins identified were considerably larger than the 12–15 kD indicated

by the markers. Thus, it was surprising to find them in this low MW band. It is

believed that because protease inhibitors were not present in our homogenization

buffer, high MW proteins might have been cleaved during the incubation period.

2.2.4 Conclusion

According to the result of the experiment using both the fluorescent PbTx-2

derivative (5) and the brevetoxin photoaffinity probe (10), it was demonstrated that

the brevetoxin is located in the chloroplast. It could be argued that the labels influence

the physiochemical properties of brevetoxin. However, these two labels have distinct

properties, yet each of these derivatives migrated to the chloroplast. The Alexa Fluor

dye, in particular, is not membrane-permeable, and the Alexa Fluor brevetoxin

conjugate would seem even less likely to enter the thylakoid membrane than native

brevetoxin. In addition, it is very clear that fluorescent PbTx-2 derivative did not label

any other organelles in the living cells. The finding that brevetoxins localize to the

lipophilic thylakoid membranes should come as no surprise. PE ladder molecules are

43

known to migrate to the cell membranes where they interact with membrane proteins.

Brevetoxins have also been demonstrated to aggregate into artificial lipid bilayers

(Woo, Liu et al.). Our findings are consistent with the localization of another

dinoflagellate toxin, okadaic acid (Zhou and Fritz 1994), to the chloroplast and the

cyanobacterial toxin, microcystin, to thylakoid area of cyanobacteria (Young,

Thomson et al. 2005)

In addition, it was determined using photoaffinity probe (10) that LHC II and Trx are

native proteins to which brevetoxin binds in K. brevis. The association with LHCII

was reduced at neutral pH or low light, conditions which were used for the initial

photoaffinity experiments. However, LHC II is highly concentrated in the thylakoids.

It is possible that our photoaffinity probe linked to LHCII simply as a result of its

high density in the thylakoids and that the true brevetoxin target in chloroplasts is a

less abundant membrane protein, such as an ion channel. Ion channels selective for

chloride, potassium, or divalent cations such as calcium and magnesium have been

found in outer- and inner-envelope membranes of chloroplasts, as well as from

thylakoid membranes, by using the patch clamp technique and have been

hypothesized to regulate photosynthesis by modulating the proton motive force

(Checchetto, Teardo et al. 2013).

In addition to LHC II, Trx is identified from both 25 kDa and 12 kDa band. It was

identified in every photoaffinity binding experiment performed. Trx is a ubiquitous

antioxidant enzyme that can be found in all living organisms from archaea to

mammals. The role of Trx is the regulation of redox homeostasis via thiol-disulfide

exchange reactions (Collet and Messens 2010) with its protein targets. It acts as a

44

reductase in redox control and protects proteins from oxidative damage (Holmgren

1985). In addition Trx helps the cells tolerate various environmental stresses, such as

ROS (Messens and Silver 2006). Numerous thioredoxin-dependent regulatory

networks have been identified in photosynthetic organisms. Thus, a better

understanding of the interaction between Trx with brevetoxin may contribute to

understanding the role of brevetoxin in K. brevis and may also have implications for

human and animal toxicity.

2.3 Materials and Method

2.3.1 Fluorescent probe experiment

Materials for fluorescent probe experiment Alexa Fluor 488; Fluorescent PbTx-2

derivative; Live K. brevis culture, a fluorescent microscope (Leica Leitz, Model

DMRB)

Method for fluorescent probe experiment

Culture growth K. brevis culture (Wilson strains) were obtained from Mote Marine

Laboratory (Sarasota, Florida) and maintained in RE medium (Sinigalliano, Winshell

et al. 2009), in a growth chamber at ~20ºC under 35 µmol.photons.m-2.s-1. Growth

was monitored by counting a 1:10 dilution of culture in Z pak reagent using a

Beckman Z-series Coulter Counter with aperture size between 10~30 µm according to

the manufacturer’s instructions.

Incubation of K. brevis with the fluorescent PbTx-2 derivative or the Alexa Fluor

488 alkyne: Live K. brevis culture (3 ml) was incubated for 1h in the dark with either

the fluorescent PbTx-2 derivative (5) or Alexa Fluor 488 alkyne (4). The final

45

concentration of the Alexa Fluor 488 alkyne was 4 µm. Because the exact

concentration of the PbTx-2 derivative was not known, the concentration of the

solution of the fluorescent PbTx-2 derivative was adjusted such that the fluorescence

was equal to that of the Alexa Fluor 488 alkyne solution. After incubation, the cells

were centrifuged for 8 min at 130 X g. The supernatant was discarded, and the cells

were resuspended in RE medium (1 ml) (Sinigalliano, Winshell et al. 2009) and

centrifuged again for 8 min at 130 X g. The supernatant was discarded, and the cells

were resuspended in RE medium (1 ml). The live cells were visualized by using a

fluorescent microscope (Leica Leitz, Model DMRB) with a 450–490 nm band

pass/515 nm long pass EX/EM filter.

2.3.2 Photoaffinity probe experiment

Materials for brevetoxin photoaffinity experiment K. brevis culture；Phosphate

buffered saline (PBS) buffer; Bovine serum albumin (BSA); Coomassie Protein

Assay Reagent (BioRad); Spinach; Photoaffinity probe (10); photoaffinity label (8);

Streptavidin beads (streptavidin immobilized on magnetic beads, Thermo Scientific

Pierce); Triton X100;

Method for brevetoxin photoaffinity experiment

Preparation of protein homogenates and membrane suspensions: The

homogenate of K. brevis cells was separated into soluble and membrane fractions. K.

brevis culture (200–400 ml) was concentrated by centrifugation for 5 min at 466 X g,

and the supernatant was discarded. The cells were resuspended in phosphate-buffered

saline (PBS, 400 µl) and vortexed for 1 min. This suspension was centrifuged for 10

46

min at 14,000 X g, and the supernatant (soluble fractions) and the pellet (membrane

fractions) were separated. The pellet was resuspended in PBS (500 µl). An aliquot (50

µl) of each fraction was analyzed by Bradford protein assay. Final protein

concentrations ranged from 0.5–0.9 µg / µl.

Spinach (5 g) was crushed in PBS (20 ml) and the biomass concentrated by

centrifugation (5 min at 14000 X g). The supernatant was discarded. The pellet was

resuspended in PBS buffer (5 ml, and an aliquot (50 µl) was reserved for Bradford

assay. The final protein concentration was 1.0–2.5 µg / µl.

Bradford assay: BSA stock slolution (30 µl, 10 mg / ml) was diluted to 300 µl (1 mg

/ ml) in PBS. Six concentrations of BSA (0 mg / ml, 1 mg / ml, 0.5 mg / ml 0.25 mg /

ml 0.125 mg / ml, 0.0625mg / ml) were prepared as standards. Commassie Protein

Assay Reagent (250 µl) was added to microplate wells. 20 µl each of standard and

unknown sample were mixed with Commassie Protein Assay Reagent in each well.

Each protein solution was assayed in duplicate. After incubation at room temperature

for 5 min, absorbance was measured at 595 nm (SynergyTM 2, Bioteck Instrument,

Inc).

Incubation of protein homogenates with photoaffinity probe or photoaffinity

label: The PbTx-2 photoaffinity probe (10) or label (8) were incubated at room

temperature with the soluble protein or membrane fractions at a probe/protein ratio of

0.2 µg probe per mg protein for 1 h. After incubation, the mixture was photolyzed

(Vilber Lourmat transilluminator, Model FLX-20M) for 20 min at 312 nm. The

soluble fraction was applied directly to a suspension of streptavidin beads (200 µl,

47

Dynabeads, 2.8 mm or 1.0 mm, Life Technologies). Triton X-100 (1 %, 200 µl) was

added to the membrane fractions (200 µl). The mixture was vortexed and centrifuged

for 5 min at 14 000 X g and applied to a suspension of streptavidin beads (200 µl).

After incubation for 30 min at room temperature, the beads were separated from the

solution by using a strong magnet and washed with PBS buffer (4 x 200 µl). Each

wash was analyzed for protein content, and the final wash contained no detectable

protein. Protein was eluted from the beads by using 0.1% SDS solution (3 x 250 µl) at

100◦C for 5 min. An aliquot of each elution was analyzed by Bradford protein assay,

concentrated to dryness by lyophilization (FreeZone, Model 2.5, 86 L/min), and

resuspended in water (10 µl) and loading buffer (2 µl, 0.25M Tris-HCl, pH 6.8, 15%

SDS, 20% glycerol, 25% mercaptoethanol, 0.01% Bromophenol Blue) for analysis by

SDS-PAGE (12% acryalamide) with Tris·glycine SDS, pH 8.3 running buffer.

Acrylamide gels were stained with PAGE Blue (Fermentas), according to the

manufacturer’s instructions. The 25 kD band was excised from the SDS-PAGE gel.

In-gel digestion: In-gel digestion and LC-ESI MS/MS analysis was performed by the

Proteomics and Mass Spectrometry Facility at the UMass Medical School. Gel slices

were cut into 1x1 mm pieces and placed in 1.5 ml Eppendorf tubes with water (1 ml)

for 30 min. The water was removed, and ammonium bicarbonate (50 µl, 250 mM)

was added. For reduction, a solution of 1,4-dithiothreitol (DTT; 5 µl, 45 mM) was

added, and the samples were incubated at 50ºC for 30 min. The samples were cooled

to room temperature and, for alkylation, iodoacetamide solution (5 µl, 100 mM) was

added and allowed to react for 30 min. The gel slices were washed with water (20 X 1

ml). The water was removed, ammonium bicarbonate/acetonitrile (50:50, 1 ml, 50

mM) was placed in each tube, and the samples were incubated at room temperature

48

for 1 h. The solution was removed, and acetonitrile (200 µl) was added to each tube,

at which point the gels slices turned opaque white. The acetonitrile was removed, and

the gel slices were further dried in a Speed Vac. Gel slices were rehydrated in trypsin

(50 µl, 2 ng / µl, Sigma) in 0.01% ProteaseMAX Surfactant (Promega) / 50 mM

ammonium bicarbonate. Additional bicarbonate buffer was added to ensure complete

submersion of the gel slices. Samples were incubated at 37 ºC for 21 h. The

supernatant of each sample was removed and placed in a separate 1.5 ml Eppendorf

tube. Gel slices were further dehydrated with acetonitrile/1% formic acid (80:20, 100

µl). The extract was combined with the supernatants of each sample. The samples

were dried in a Speed Vac. Samples were dissolved in 25 µl of 5% acetonitrile in 0.1%

trifluoroacetic acid prior to injection on LC-MS/MS.

LC-MS/MS on Q Exactive: For peptide sequencing, a 3.0 µl aliquot was directly

injected onto a custom-packed 2 cm X 100 mm C18 Magic 5 m particle trap column.

Labeled peptides were eluted and sprayed from a custom-packed emitter (75 µm X 25

cm C18 Magic 3 µm particle) with a linear gradient from 95% solvent A (0.1% formic

acid in water) to 35% solvent B (0.1% formic acid in acetonitrile) over 40 min at a

flow rate of 300 nL/min on a Waters Nano Acquity UPLC system. Data dependent

acquisitions were performed on a Q Exactive mass spectrometer (Thermo Scientific),

according to an experiment in which full MS scans from 300–1750 m/z were acquired

at a resolution of 70,000, followed by 12 MS/MS scans acquired under higher-energy

collisional dissociation (HCD) fragmentation at a resolution of 35 000 with an

isolation width of 1.2 Da.

49

Database searching: Raw data files were processed with Proteome Discoverer (v.

1.3). Charge state deconvolution and deisotoping were not performed. All MS/MS

samples were analyzed by using Mascot (Matrix Science, London, UK; v. 2.4.1).

Mascot was set up to search the NCBInr_20130907 database (32265160 entries),

assuming the digestion enzyme strict trypsin. Mascot was searched with a fragment

ion mass tolerance of 0.050 Da and a parent ion tolerance of 10.0 ppm. The

carbamidomethyl of cysteine was specified in Mascot as a fixed modification.

Gln >pyro-Glu of the N terminus, oxidation of methionine, and acetylation of the N

terminus were specified in Mascot as variable modifications.

Criteria for protein identification: Scaffold (v. 4.1.1, Proteome Software Inc.,

Portland, OR) was used to validate MS/MS-based peptide and protein identifications.

Peptide identifications were accepted if they could be established at greater than 80.0 %

probability by the Peptide Prophet algorithm (Keller, Nesvizhskii et al. 2002) with

Scaffold delta-mass correction. Protein identifications were accepted if they could be

established at greater than 90.0% probability and contained at least two identified

peptides. Protein probabilities were assigned by the Protein Prophet algorithm

(Nesvizhskii, Keller et al. 2003). Proteins that contained similar peptides and could

not be differentiated based on MS/MS analysis alone were grouped to satisfy the

principles of parsimony. Proteins sharing significant peptide evidence were grouped

into clusters.

50

Chapter 3 The effects of brevetoxins on the thioredoxin reductase/thioredoxin

system and related enzymes.

3.1 Objective

The findings described in the previous chapter indicated that brevetoxin binds to Trx.

This finding promoted us to examine how brevetoxin affects the TrxR/Trx system.

Because Trx is present in all living organisms and is key to regulation of the activity

of numerous enzymes and enzyme systems, this work can contribute to our

understanding of the key role of berevetoxin in K. brevis cells and mechanism of

toxicity to other organisms.

3.2 Introduction

Trx is the parent of a family of enzymes which share structural (but not necessarily

sequence) homology and function through thiol-disulfide exchange. The thioredoxin

superfamily is responsible for maintenance of the redox state within cells through the

reduction of thiol disulfide bridges. The thioredoxin superfamily includes the Trx 1

and Trx 2 (Trx A and Trx C) in the cytoplasm (Miranda-Vizuete, Damdimopoulos et

al. 1997, Stewart, Åslund et al. 1998) and glutaredoxins 1, 2, 3 (GrxA, GrxB, GrxC)

(Aslund, Ehn et al. 1994). A number of eukarytoic proteins such as protein disulfide

isomerase (PDI) also belong to the thioredoxin superfamily (Carvalho, Fernandes et

al. 2006). Some bacterial proteins like E.coli DsbA (a bacterial thiol disulfide

oxidoreductase) also contains thioredoxin domains (Carvalho, Fernandes et al. 2006).

Although those proteins do not all share much similarity in sequence, all proteins

have a common sequence CXXC motif in the active site (Table 5). This motif is a

highly conserved region which presents at the end of an α–helix in all proteins. The

amino acids between these two cysteines vary within the thioredoxin superfamily

51

(Carvalho, Fernandes et al. 2006). In thioredoxin the sequence is CGPC, CPYC in

glutaredoxins, CPHC in PDIs, and CGHC in DsbA.

Table 5 The motifs in thioredoxin , glutaredoxin, PDI and DsbA (Holmgren, Söderberg et al. 1975)

Enzyme CXXC motif Function Thioredoxin CGPC Reductant Glutaredoxin CPYC Reductant

PDI CHGC Oxidant Isomerase DsbA CPHC Oxidant

Trx catalyzes the disulfide reduction as a bimolecular nucleophilic substitution

reaction (SN2). During this process, the disulfide bond of the target is transferred to

Trx. In other words, the substrate protein acquires the electrons from Trx and Trx

becomes oxidized to the disulfide. The N-terminal cysteine of this CXXC motif is

deprotonated under physiological conditions and exposed to the aqueous

surroundings. This N-terminal cysteine of the motif serves as the nucleophilic

cysteine. The other cysteine in the motif is usually buried in the enzyme and

protonated (Carvalho, Fernandes et al. 2006). In all enzymes, the nucleophilic

cysteine must be deprotonated to participate in thiol-disulfide exchange. Typically, a

disulfide bond of the substrate is attacked by the N-terminal cysteine thiolate to form

a mixed disulfide bond (Holmgren 1995, Jeng, Reymond et al. 1998). The thiolate on

the C-terminal cysteine is stabilized with two backbone amide hydrogen bonds,

lowering the pKa (Roos, Foloppe et al. 2009). N-terminal cysteine is attacked by C-

terminal cysteine thiolate to form the Trx disulfide. A reduced substrate protein and

oxidized Trx are the products of this exchange reaction (Figure 15).

52

Figure 15 Trx catalyzes the reaction as a bimolecular nucleophilic substitution reaction (SN2)

The reaction between Trx and its substrate is stoichiometric. Oxidized Trx is more

stable than reduced Trx (Collet and Messens 2010). It has been proposed that the rate

of the thiol- disulfide exchange is affected by several factors, such as the pKa of the

nucleophilic cysteine (Shaked, Szajewski et al. 1980), the local electrostatic

environment of the nearby amino acid (Bulaj, Kortemme et al. 1998, Hansen,

Østergaard et al. 2005), the molecular strain (Østergaard, Henriksen et al. 2001), the

pH of the surrounding solvent (Setterdahl, Chivers et al. 2003) and the pKa values of

the leaving thiol groups of the oxidized substrate (Shaked, Szajewski et al. 1980,

Jensen, Hansen et al. 2009).

Over the past 50 years, Trx has been studied extensively. The first Trx was discovered

in 1964 in E. coli as an electron donor to ribonucleotide reductase, an essential

enzyme for DNA synthesis (Laurent, Moore et al. 1964, Moore, Reichard et al. 1964).

Since then, Trx has been determined to play multiple cellular roles, such as reductase

in redox control (Holmgren 1985), protecting proteins from oxidative damage and

inactivation (Holmgren 1985, Holmgren 1989, Holmgren 1995) and helping the cells

to combat various environmental stresses (reactive oxygen species (ROS),

peroxynitrite, arsenate) (Landino, Skreslet et al. 2004). Some Trxs can also regulate

cell death via denitrosylation and modulate the inflammatory response (Rahman and

Adcock 2006). In addition, it has been shown that Trx plays an essential role in the

life cycle of viruses. Trxs are located in various cellular compartments. For instance, a

53

number of Trxs are found in the cytosol (Arnér and Holmgren 2000), while others are

abundant in nucleus or mitochondria (Hirota, Matsui et al. 1997, Hirota, Murata et al.

1999). Still others are associated with the cell membrane (Martin and Dean 1991).

In 1964 the Trx/TrxR system was determined to be comprised of Trx, together with

thioredoxin reductase (TrxR) and NADPH (Laurent, Moore et al. 1964). In general,

the electrons which are provided by NADPH are transferred to the active site of Trx

via TrxR. The disulfide of oxidized Trx must be reduced back to a dithiol to regain its

active form. The enzyme TrxR, with reducing equivalents ultimately derived from

NADPH (Figure16) performs this reduction.

Figure 16 Electron flow of E. coli TrxR/Trx system

TrxR is a member of the pyridine nucleotide disulfide oxidoreductase family that

includes glutathione reductase (GR) and lipoamide dehydrogenase (LipDH)

(Mustacich and Powis 2000). Both TrxR and GR use NADPH as a cofactor via FAD

to transfer electrons to the redox centers, while LipDH uses NADH instead of

NADPH (Williams Jr 1992). In addition, two types of TrxR have been identified.

Although they both have same functions, the amino acid sequence and catalytic

mechanisms are distinct (Gromer, Schirmer et al. 1999, Mustacich and Powis 2000).

The type of TrxR with a low molecular weight (~35 kDa) has been characterized in

54

Archaea, bacteria and Eukarya (Becker, Gromer et al. 2000). There are two binding

domains in E. coli TrxR: FAD binding domain and NADPH binding domain. The

connection of these two domains is not close (Williams 1995). In E. coli TrxR the

FAD domain is rotated 66 degrees with NADPH domain remaining, so that the two

prosthetic groups move into close contact. The conformational change allows

electrons to pass from NADPH to FAD then to reduce the disulfide bond (Lennon and

Williams 1997, Mustacich and Powis 2000). The conserved active site residues are –

Cys-Ala-Thr-Cys- in the redox center of E. coli TrxR.

A high molecular weight (~55 kDa) TrxR is often referred to as mammalian as it was

first identified in Homo sapiens. Subsequently, this high molecular weight TrxR has

been found to be widespread in the animal kingdom (Mustacich and Powis 2000,

Bauer, Gromer et al. 2003). Mammalian TrxR is distinct from bacterial TrxR by

virtue of a third redox center in a C-terminal extension of the enzyme consisting of a

cysteine and a selenocysteine (Sec) (Cys497-Sec498-Gly- in human TrxR). The

purification of rat liver Trx and TrxR demonstrated that mammalian TrxR is larger

than mammalian Trx. Mammalian TrxR has broader substrate specificity than

prokaryotic forms (Luthman and Holmgren 1982, Arnér 2009). Mammalian TrxR is a

large selenoenzyme, which has three isoforms: TrxR1 in the cytosol (Arnér 2009),

TrxR2 in mitochondria and TrxR3 or thioredoxin glutathione reductase (TGR)

(testis)(Lillig and Holmgren 2007). TGR has common properties to other flavoprotein

oxidoreductase. However, TGR is the unique enzyme that connects thioredoxin and

glutathione system. TGR has an extra reaction sites (CVNVGC) at the N-terminal.

The reducing equivalents are transferred from NADPH to the FAD. Then, the

adjacent cysteine in the reaction site (CVNVGC) at C-terminal. The reduced cysteine

55

transfers the hydride to the cysteine-selenocysteine in the C-terminal reaction center

(GUCG) of the other peptide chain (Figure17 A). The reduced GUCG produces a

structural change allowing to reduce either extra active site (CPYC) of TGR or

oxidized thioredoxin. The reduced active site (CPYC) interacts with glutathione

disulfide (Figure 17 B).

Figure 17 Reaction mechanism of TGR. (A) The reducing equivalents are transferred from NADPH to the FAD. Then, the adjacent cysteine in the reaction site (CVNVGC) at C-terminal. The reduced cysteine transfers the hydride to the cysteine-selenocysteine in the C-terminal reaction center (GUCG) of the other peptide chain. (B) The reduced GUCG produces a structural change allowing to reduce either extra domain (CPYC) of TGR or oxidized thioredoxin. The reduced extra domain interacts with oxidized glutathione

56

It has been found that the selenium is the essential active site residue in mammalian

TrxR. Blocking or mutation at the selenium active site results in a loss of ability to

reduce Trx (Figure18).

Figure 18 Electron flow of mammalian TrxR/Trx system The Trx/ TrxR system plays a role as a general protein disulfide reductase. It is

involved in regulating DNA synthesis, gene transcription, cell growth and metabolism

(Krauth-Siegel and Coombs 1999, Arnér and Holmgren 2000, Mustacich and Powis

2000, Williams 2000). The Trx/TrxR system also plays a key role in biochemical

mechanisms associated with many human diseases (Figure19). For instance, Trx and

TrxR overexpression is observed in many cancer cells, which has been hypothesized

to be a protection against apoptosis (Baker, Payne et al. 1997, Powis, Mustacich et al.

2000, Lincoln, Ali et al. 2002, Urig and Becker 2006, Anestål, Prast-Nielsen et al.

2008). TrxR is an important target for many cancer treatments. Cisplatin and arsenic

trioxide as clinical drugs are used to inhibit TrxR resulting in blocking or mediating

the activity of DNA synthesis and defense against oxidative stress (Lu, Chew et al.

2007, Arnér 2009).

57

Figure 19 The Trx/TrxR system plays a key role in biochemical processes associated with many human diseases

In most organisms in addition to Trx/TrxR system, glutathione/ glutathione disulfide

(GSH/GSSG) is a major pathway for regulating cellular redox homeostasis and

antioxidant defense. The mammalian GSH/GSSG system involved GSH recycling of

oxidized GSSG catalyzed by GR using NADPH as electron donor The N-terminal

domains of GR resemble those of TrxR , including the FAD prosthetic group, the

NADPH binding domain and the N-terminal redox-active CVNVGC motif (Arscott,

Gromer et al. 1997). However, GR lacks the C-terminal redox center (GCUG) of

TrxR (Lu and Holmgren 2014).

In addition to Trx, Grx and glutathione peroxidase (GPx) also belong to the family of

oxidoreductases which function through thiol-disulfide exchange. These proteins have

similar three-dimensional structures and contain an active site dithiol/disulfide group

which protrudes from a hydrophobic protein surface The disulfide bond in Grx is

reduced continually by GSH in the mammalian GSH/GSSG system (Figure 20).

While Trx and Grx show low sequence homology, they exhibit similar three-

58

dimensional topology including the so-called thioredoxin fold, consisting of an N-

terminal βαβ motif and a C-terminal ββα motif with the central β-sheets surrounded

by the α-helices and an active site CXXC motif, which protrudes from the

hydrophobic surface of the protein.(Martin 1995, Netto, de Oliveira et al. 2016)

. Figure 20 Electron flow of mammalian GSH/GSSG system

GPx catalyzes the reduction of ROS in cells, including hydrogen peroxide, by reduced

glutathione to protect the cells from oxidative damage. Moreover, most noteworthy is

that GPx is a selenoprotein. GPx has four identical subunits. Each subunit contains a

selenocysteine in the active site, which participates directly within the catalytic cycle.

The selenium undergoes a redox cycle involving the selenol (GPx-SeH) as an active

site to reduce hydrogen peroxides and other harmful peroxide, which oxidizes GPx-

SeH to selenenic acid (GPx-SeOH). A reduced GSH reacts with GPx-SeOH to form

selenenyl sulfide adduct (GPx-SeSG). A second GSH is the ultimate electron donor to

regenerate the reduced form of the selenocysteine by attacking the GPx-SeSG to form

the oxidized GSSG (Figure21) (Morgenstern 2005, Toppo, Vanin et al. 2008).

59

Figure 21 The redox cycle of GPx coupled with mammalian GSH/GSSG system

3.3 Results

3.3.1 Modified papain assay

The modified papain assay is a sensitive spectrophotometric assay that is commonly

used for the quantitation of thiol groups. The assay involves the reaction of a thiol

with an inactive mixed disulfide of papain, a cysteine protease, resulting in the

stoichiometric formation of active papain (Figure 22) (Singh, Blattler et al. 1993). The

active site cysteine of papain is converted to an inactive mixed disulfide which may

be reduced by low MW thiols to the active form. The active papain then catalyzes the

hydrolysis of N-benzoyl-L-arginine-p-nitroanilide to yield p-nitroanilide.

Figure 22 The equation of modified papain assay

The chromogenic product can be detected at 412 nm. Furthermore, the addition of the

disulfide cystamine to the assay allows for the detection of inaccessible protein thiols.

Cystamine is reduced to cysteamine by protein thiols and the resulting cysteamine

acts as a shuttle between high MW inaccessible protein thiols and modified papain.

60

Thus the difference in papain activity with and without cystamine is a reflection of

high MW inaccessible thiols. The initial amount of thiol can be obtained from the

proportion of absorbance compared to the concentration of thiol standards. Here, this

method can also be used to determine the content of reduced Trx. Because the

reaction of Trx with it’s substrates is stoichiometric, the modified papain assay can be

used to amplify the signal of Trx. Trx is used to reduce the small disulfide cystamine

to cysteamine, which will in turn reduce the papain disulfide stoichiometrically

(Figure23). Thus the modified papain assay may be used to assay for Trx activity by

amplification of the Trx signal.

Figure 23 The principle of modified papain assay to determine Trx activity

3.3.2 Papain assay

The effect of PbTx-2 on Trx was examined using the modified papain assay.

Inhibition of this two enzyme (Trx/papain) system was observed in the presence of

PbTx-2 (15 µM) when compared to the positive control reaction (no PbTx) whether

the PbTx-2 was pre-incubated with Trx or not pre-incubated (Figure24).

61

Figure 24 The modified papain assay with / without PbTx-2 The condition: PbTx-2 (15 µM), substrate (1 mM), modified papain (10 µM), cystamine (45 µM), Trx (6 µM), pH 7.0 acetate buffer (50 mM)

However, comparison of the relative initial rates (V0) for the pre-incubated and non-

pre-incubated reactions, shows that pre-incubation with Trx results in a 56%

reduction of V0, compared to a 36% reduction of V0, observed without pre-incubation.

Thus pre-incubation of Trx with PbTx-2 resulted in increased inhibition of Trx.

Because pre-incubation of Trx and PbTx-2 increased the inhibition in the modified

papain assay, this suggests that PbTx-2 does indeed act as an inhibitor of Trx.

However, there are two complicating factors which need to be considered: first, the α,

β-unsaturated aldehyde side chain of PbTx-2 can react as an electrophile in a Michael

addition, with the generated cysteamine and second, it has been reported both PbTx-2

and -3 are inhibitors of papain (Sudarsanam, Virca et al. 1992). To address the first

complication, another papain assay was used PbTx-2 as inhibitor but without

cystamine. PbTx-2 does show inhibition in the absence of cystamine, but under these

conditions the reaction is slow and the assay is much less sensitive (Figure25).

62

Figure 25 The modified papain assay with / without PbTx-2 but without cystamine. The condition: PbTx-2 (15 µM), substrate (1 mM), modified papain (10 µM), Trx (6 µM), pH 7.0 acetate buffer (50 mM)

Thus, the rate of the reaction in the presence of Pbtx-3 (which does not react with

cysteamine) was determined to see if the effect of brevetoxin is due to reaction with

cysteamine. It is clear that the Pbtx-3 impacts the rate (Figure 26).

Figure 26 The modified papain assay with / without PbTx-3 with cystamine. The condition: Pbtx-3 (15 µM), substrate (1 mM), modified papain (10 µM), cystamine (45 µM), Trx (6 µM), pH 7.0 acetate buffer (50 mM)

In order to understand the inhibition of papain by Pbtx-2 and Pbtx-3, a standard

papain assay which didn’t contain the cystamine was performed in the presence of

PbTx-2 and PbTx-3 (Figure27). Figure 27 shows that both brevetoxins inhibit papain.

63

Figure 27 The papain assay with / without brevetoxin. The condition: PbTx-3 or PbTx-2 (15 µM), substrate (1 mM), papain (10 µM), MeOH 3.2%, pH 7.0 acetate buffer (50 mM) The initial rates (V0) are calculated based on the polynomial regression of the trend

lines in Figure 24 26 and 27. Table 6 compares initial rates for the papain and

modified papain assays with and without inhibitor relative to the standard papain

assay without inhibitor. The initial rate (V0) of the standard papain assay is three fold

higher than that of the modified papain assay. If inhibition of the modified papain

assay by brevetoxin is due solely to the inhibition of papain, one would anticipate a

similar reduction in V0 in the presence of brevetoxin. However, a six fold reduction of

V0 was shown in the presence of brevetoxins (PbTx-2 and PbTx-3) for the modified

papain assay. Table 7 shows that the addition of PbTx-3 (15 µM) results in a 15%

reduction of V0 in the standard papain assay when compared to the control. However,

when added to the modified papain assay under the same conditions, a 52% reduction

of V0 is observed. While PbTx-2 (15 µM) results a 25% reduction of V0 in the

standard papain assay whereas it results in a 56% reduction of V0 in the modified

papain assay. These data suggest that brevetoxins are indeed inhibitors of Trx.

However, it still cannot directly prove that the inhibition is caused by brevetoxin.

Table 6 Relative V0 for the assays shown above in Figure 24,26 and 27

Inhibitor Only papain

Modified Papain assay

Fold reduction

None 1 0.32 3.1

64

Brevetoxin-2 (15 µM) 0.75 0.12 6.3 Brevetoxin-3 (15 µM) 0.85 0.15 5.6

Table 7 The inhibition % for the assays shown above in Fig 24,26 and 27

Only papain Modified papain assay PbTx-2 25% 56% PbTx-3 15% 52%

3.3.3 Fluorescent assay for Trx/TrxR system

As an alternative, the activity of Trx can be amplified at the front end by including

TrxR and NADPH in the reaction to continuously reduce the Trx. The method used is

based on the reduction of insulin by reduced Trx with NADPH as ultimate electron

donor via TrxR (Montano, Lu et al. 2014). The insulin is labeled with fluorescein

isothiocyanate-labeled (FiTC). These FiTC-modified insulin servers as the substrate.

Insulin is composed of two peptide chains which are the chain A and chain B. It is

found that the fluorescence is quenched by the disulfides between chain A and B.

However, the fluorescence is increased on reduced form of insulin (Heuck and

Wolosiuk 1997). Here, the disulfides are able to be reduced by Trx /TrxR system, the

fluorescence of FiTC is record after 30 min incubation with the mixture of Trx, TrxR

and NADPH (Figure 28) (Montano, Lu et al. 2014).

Figure 28 The reaction in sensitive fluorescent assay for Trx/TrxR system

To determine if brevetoxin acts as inhibitor of Trx, the inhibition of Trx by PbTx-2

and -3 was determined using a commercially available assay kit (Montano, Lu et al.

2014). PbTx-2 and PbTx-3 at concentrations ranging from 10 to 35 µM were

incubated with a mixture of pre-reduced TrxR and Trx for one hour. After adding

65

FiTC-modified insulin substrate, the release of fluorescent reporter was monitored.

The first 40 minutes correspond to a slow induction period where two disulfide

bridges of Trx are reduced and the increase in fluorescence is not linear. Therefore,

the data for the first 40 minutes is not used in accordance with the manufacturer’s

instructions. The rate of reaction within the linear range was determined by using the

slopes of the linear trend lines. A graph was made by using the concentration of

PbTx-2 as x-axis and the resulting values of each corresponding curve lines as y-axis

(Figure 29.) IC50 was calculated as the concentration of an inhibitor that is required

for 50% inhibition. Interestingly, PbTx-2 exhibited a dose dependent inhibition of

Trx/TrxR activity with an IC50 of 25 µM, whereas PbTx-3 had no effect on the rate of

insulin reduction (Figure 30). It has been shown that apparent Km values for rat Trx

3.7 µM (Heuck and Wolosiuk 1997). Based on Cheng-Prusoff equation, Ki was

determined at 24.7 µM.

Figure 29 The effect of PbTx-2 on Trx/TrxR system. A: Rat TrxR and human Trx pre-reduced with NADPH (0.35 mM) for 30 min followed by incubation with brevetoxins for 1 h prior to addition of insulin substrate. B: The concentration of PbTx-2 vs the initial rate of reaction. IC50 was calculated as the concentration of an inhibitor that is required for 50% inhibition. PbTx-2 exhibited a dose dependent inhibition of Trx/TrxR activity with an IC50 of 25 µM

A

B

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Figure 30 The effect of PbTx-3 on Trx/TrxR system. A: Rat TrxR and human Trx pre-reduced with NADPH (0.35 mM) for 30 min followed by incubation with brevetoxins for 1 h prior to addition of insulin substrate. B: The concentration of PbTx-2 vs the initial rate of reaction. However, PbTx-3 had no effect on the rate of insulin reduction

3.3.4 Ellman’s assay

Ellman’s assay utilizes 5, 5’-dithio-bis-(2-nitrobenzoic acid), also known as DTNB, to

react with free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic

acid (TNB). TNB is a measurable yellow-colored product (Figure 31) which can be

detected at 412 nm (Riener, Kada et al. 2002). To detect the Trx or TrxR activity,

reduced Trx or TrxR can be used to reduce DTNB producing the yellow TNB which

can be detected at 412 nm.

Figure 31 The reduction of Ellman’s reagent

Because the Trx/TrxR assay is a two enzyme system (Trx/TrxR), it was still not clear

if the inhibition observed in the presence of PbTx-2 is due to the inhibition of Trx, or

TrxR or both. Unlike bacterial TrxR, which shows high substrate selectivity for Trx,

mammalian TrxR reduces a broad range of substrates, including small disulfides such

as DTNB, making it possible to assay for TrxR activity in the absence of Trx. Thus,

A

B

67

the activity of TrxR alone in the presence and absence of PbTx-2 was determined by

using Ellman’s reagent.

PbTx-2 (22 µM) was incubated with the rat TrxR in the presence of NADPH. After

30 min, DTNB was added to the reaction mixture and the absorbance of TNB- was

monitored for 60 mins. Surprisingly, the Vo of DTNB reduction by TrxR was

enhanced 2.5 fold in the presence of PbTx-2 when compared to the control reaction

which lacked PbTx-2. (Figure32 A). The same result was obtained when PbTx-2 was

added simultaneously with DTNB. In other words, without pre-incubation of TrxR by

NADPH and PbTx-2. (Figure32 B)

Figure 32 The effect of PbTx-2 on the Ellman’s assay. (A) The effect of PbTx-2 on the DTNB reduction by pre-incubated TrxR. Rat TrxR was pre-reduced with NADPH (0.10 mM) for 30 min with PbTx-2 (18 µM), followed by addition of DTNB (1.8 mM). (B) The effect of PbTx-2 on the DTNB reduction by non-incubated TrxR. Rat TrxR was pre-reduced with NADPH (0.10 mM) for 30 min without PbTx-2 (18 µM), followed by addition of DTNB (1.8 mM)

That surprising result was reproducible and results triggered our curiosity: Why does

PbTx-2 enhance the activity of TrxR?

As mentioned earlier, mammalian TrxR is distinct from bacterial TrxR by virtue of a

third C-terminal redox center consisting of a cysteine and a selenocysteine (Sec or U).

The TrxRs which were used in all experiments to this point were mammalian. In the

A

B

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enzymatic mechanism of Trx reduction by TrxR, NADPH provides the reducing

equivalents and initiates this cascade of redox reactions across three redox active sites:

FAD which is first reduced by NADPH, next FADH2 reduces an N-terminal disulfide

(in human TrxR:- Cys59-Val-Asn-Val-Gly-Cys64) which undergoes thiol disulfide

exchange with a C-terminal disulfide or seleno-sulfide (in human TrxR:-Gly-Cys497-

Sec498-Gly). The C-terminal redox center of TrxR undergoes thiol-disulfide exchange

with oxidized Trx (Figure 33).

Figure 33 The electron flow in C-terminal reaction center in mammalian TrxR

Furthermore, it is reported the activity of Sec is essential for mammalian TrxR to

catalyze the reduction of Trx (Lee, Bar-Noy et al. 2000, Zhong and Holmgren 2000).

When the Sec site is blocked or modified, the mammalian TrxR is unable to reduce

the oxidized Trx disulfide to activated Trx-(SH)2. Sec (U) is a selenium containing

analog of cysteine but with a stronger nucleophile, which makes Sec a highly reactive

amino acid. Thus many compounds such as quinol (Bradshaw, Matthews et al. 2005),

curcumin (Fang, Lu et al. 2005) and 4-hydroxy-2-nonenal (Fang and Holmgren

2006)(Figure34) inhibit TrxR as electrophilic compounds. These molecules can

69

function as electrophiles, or Michael acceptors, for the nucleophilic Cys or Sec

residues, resulting in irreversible inhibition of TrxR. Curcumin was found to alkylate

both the C-terminal cysteine and selenocysteine of TrxR. On the other hand, 4-

hydroxy-2-nonenal and quinols have been demonstrated to label the C-terminal

cysteine and selenocysteine of TrxR as well as the redox active cysteines of Trx.

Figure 34 The Michael acceptors demonstrated to inhibit TrxR Comparison of these compounds to PbTx-2, reveals that they have a common

functional group: an α, β-unsaturated carbonyl. Thus, it is assumed that the α, β-

unsaturated aldehyde K-ring side chain of PbTx-2 may function as an efficient

electrophile, reacting with Sec. Thus, if the TrxR and PbTx-2 form an adduct, this

would result in the irreversible inhibition of Trx reduction, which can explain the

reason PbTx-2 shows inhibition of the TrxR/Trx system towards insulin reduction

while the allylic alcohol, PbTx-3 does not (Figure35). This explanation is also

consistent with the observation that DTNB reduction by TrxR is actually enhanced in

the presence of PbTx-2. It has been previously demonstrated that small molecules

such as DTNB can be reduced by the N-terminal redox center while the C-terminal

center is alkylated (Cenas, Nivinskas et al. 2004, Eckenroth, Lacey et al. 2007). In the

reduction of Trx by TrxR, is has been shown that the reduction of the C-terminal

seleno-sulfide is the rate-determining step. Reduction of DTNB by the N-terminal

70

disulfide actually by passes the rate-determining step. Thus, the activity of TrxR

towards the reduction of DTNB was increased (Figure 36).

Figure 35 The hypothesized explanation for the observation in fluorescent Trx/TrxR assay with PbTx-2

Figure 36 The hypothesized explanation for the observation in DTNB assay with TrxR and PbTx-2 In order to support this hypothesis, we were able to obtain a truncated recombinant

human TrxR which lacks the C-terminal Sec residue (Cheng, Sandalova et al. 2009,

Lothrop, Ruggles et al. 2009, Snider, Dustin et al. 2014). However, truncated TrxR,

which lacks the C-terminal Sec residue retains its ability to reduce low molecular

weight disulfides via its N-terminal dithiol redox center. In order to identify the redox

71

center which is affected by brevetoxin, the activity of truncated human TrxR was

examined in the presence of PbTx-2. At 22 µM, PbTx-2 had no effect on the activity

of truncated hTrxR towards the reduction of DTNB when compared to the control

sample, which lacked PbTx-2 (Figure 37). This suggests that PbTx-2 interacts with

the C-terminal redox center of TrxR.

Figure 37 The effect of truncated recombinant human TrxR by PbTx-2. Truncated human TrxR was pre-reduced with NADPH (0.10 mM) for 30 min in the presence of PbTx-2 (18 µM), followed by DTNB (1.8 mM)

The results from all experiments support our hypothesis that PbTx-2 inhibits TrxR by

forming an adduct with the C-terminal Sec residue. However, direct evidence for

adduct formation between the α, β-unsaturated aldehyde K-ring side chain of PbTx-2

with TrxR was still lacking at this point. In order to confirm the hypothesis, PbTx-3

was used, which only has alcohol instead of aldehyde to react the mammalian TrxR

under the exactly same condition. Interestingly, the rate of DTNB reduction by TrxR

was also enhanced in the presence of PbTx-3 but only 1.31 fold (Figure38). However,

it could be many reasons to explain the observations. For example, perhaps

brevetoxin binding can induce a conformational change in TrxR. Also, perhaps the α,

β-unsaturated unsaturated carbonyl in the A ring lactone may actas a Michael

acceptor, although less effectively than the α, β-unsaturated unsaturated aldehyde of

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PbTx-2

Figure 38 The effect of PbTx-3 on DTNB reduction by mammalian TrxR. Rat TrxR was pre-reduced with NADPH (0.10 mM) for 30 min with PbTx-3 (18 µM), followed by addition of DTNB (1.8 mM) 3.3.5 Adduct formation between PbTx-2 and selenocysteine

The demonstration of adduct formation between PbTx-2 with selenocysteine could

provide strong evidence to prove PbTx-2 is able to react with mammalian TrxR. Thus

seleno-L-cysteine was incubated with PbTx-2 (2:1) overnight. Two prominent peaks

were revealed in mass spectrum (m/z = 1080.44355) corresponding to [M + H] and

(m/z = 1102.42563) corresponding to [M + Na] for C53H77NO17Se, an oxidized adduct

of PbTx-2 and selenocysteine. An m/z for PbTx-2 (894. 4766) was not observed,

indicating the reaction was quantitative for PbTx-2 (Figure39). The predicted Se

isotopic ratio of 76Se(9%),77Se(8%),78Se(24%),80Se(50%),82Se(9%) was observed in

both products (Ip, Birringer et al. 2000) ( Table 8).

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Figure 39 The mass spectrum of adduct formation between PbTx-2 with selenocysteine. Two prominent peaks were revealed in mass spectrum (m/z = 1080.44355) corresponding to [M + H] and (m/z = 1102.42563) corresponding to [M + Na] for C53H77NO17Se, an oxidized adduct of PbTx-2 and selenocysteine. An m/z for PbTx-2 (894. 4766) was not observed, indicating the reaction was quantitative for PbTx-2. Acknowledge to Freddy Rodriguez

Table 8 The predicted Se isotopic ratio

Isotopic Se [M+H] [M+Na]

76Se 1076 1098 77Se 1077 1099 78Se 1078 1100 80Se 1080 1102 82Se 1082 1104

3.3.6 Adduct formation between PbTx-2 and TrxR

Although the previous experiment demonstrated that PbTx-2 is capable of forming an

adduct with selenocysteine, it is not a direct evidence to confirm that mammalian

TrxR is alkylated by PbTx-2. In an effort to obtain direct evidence of adduct

formation between PbTx-2 and TrxR, reduced TrxR was incubated overnight with a

20-fold excess of PbTx-2. Samples were analyzed by SDS PAGE and the bands of

anticipated size were excised from the gel and subjected to in-gel trypsin digestion,

treatment with iodoacetamide (IAC) followed by MS/MS analysis. MS/MS analysis

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of the peptides from the control sample (TrxR only and no PbTx-2) revealed the

presence of numerous peptides of predicted size according to the amino acid sequence

and the anticipated IAC modified fragments (Fig 40). These included the predicted C-

terminal peptide SGGDILQSGCUG, doubly charged and dialkylated with IAC at

both the cysteine and selenocysteine residues. From the PbTx-2 incubated TrxR

sample, the anticipated PbTx-2 modified C-terminal peptide SGGDILQSGCU(PbTx-

2)G was not observed (Figure41). Nonetheless, in the PbTx-2 treated TrxR, the

peptide sequence SGGDILQSGCUG either modified with IAC or unmodified was

conspicuously absent. Because it has been published that when the enzyme is

oxidized, it is resistant to carboxypeptidase or trypsin treatment (Zhong, Arnér et al.

1998). In our case, DTT was used to reduce all disulfide bonds before trypsin

treatment. However, the peptide sequence SGGDILQSGCUG was still un-observable.

It is possible that PbTx-2 alkylated TrxR is resistant to trypsin digestion.

Figure 40 MS/MS analysis of the peptides from rat liver TrxR. Samples were analyzed by SDS PAGE and the bands of anticipated size were excised from the gel and subjected to in-gel trypsin digestion, treatment with iodoacetamide (IAC) followed by MS/MS analysis. The peptides of predicted size according to the amino acid sequence were highlighted by yellow. The anticipated IAC modified fragments were highlighted by green. The predicted C-terminal peptide SGGDILQSGCUG, doubly charged and dialkylated with IAC at both the cysteine and selenocysteine residues (last row)

Figure 41 MS/MS analysis of the peptides from the adduct formation between PbTx-2 and TrxR. Samples were analyzed by SDS PAGE and the bands of anticipated size were excised from the gel and subjected to in-gel trypsin digestion, treatment with iodoacetamide (IAC) followed by MS/MS analysis. The peptides of predicted size according to the amino acid sequence were highlighted by yellow. The anticipated PbTx-2 modified C-terminal peptide SGGDILQSGCU(PbTx-2)G was not observed. Nonetheless, in the PbTx-2 treated TrxR, the

75

peptide sequence SGGDILQSGCUG either modified with IAC or unmodified was conspicuously absent (last row) 3.3.7 Reaction of TrxR with Sel-green probe

The selenol selective probe, Sel-green (11) was prepared as previously reported

(Zhang, Ge et al. 2015).The Sel-green probe undergoes a nucleophilic aromatic

substitution (SNAr) reaction selectively with selenols to release a fluorescent

coumarin reporter (Figure 42). After incubation of reduced TrxR with an 8 fold

excess of PbTx-2, Sel-green was added and the fluorescence increase was monitored

(λex/em = 370/502 nm). The addition of the Sel-green probe, to reduced mammalian

TrxR resulted in release of the fluorescent reporter. If Sec is alkylated by PbTx-2,

then the fluorescence reporter should not be released in the presence of PbTx-2 and

fluorescence should not be observed. We anticipated a 1:1 stoichiometry the reaction

between TrxR and the Sel-green probe and while the concentration of TrxR (5.3 µM

based on FAD absorbance) is below the limit for accurate quantitation for the

reporter, the fluorescence in the absence of PbTx-2 is comparable to the fluorescence

released in the presence of selenocysteine (6 µM) as a control. Fluorescence is clearly

reduced in the presence of PbTx-2 to a level that is only slightly higher than the

control sample which doesn’t contains PbTx-2 (Figure 43).

Figure 42 Reaction of Sel-Green probe with selenocysteine

NO2O2N

SHN

O OO

O

N

ON

NH2

O NO2

O2N

SeCO2H

H2NSec

SO2

Fluorescent ReporterSel-green 11

76

Figure 43 Release of fluorescent reporter from Sel-green probe upon incubation with TrxR in the presence and absence of PbTx-2 compared to standards of L- selenocysteine (3 µM and 6 µM). Rat TrxR (5.3 µM ) was incubated with sel-green probe (43 µM ) with presence and absence of PbTx-2 (48 µM ). Control experiment: Seleno-L-cysteine (3 µM and 6 µM ) was incubated with sel-green probe ( 43 µM ) 3.3.8 Grx, GPx fluorescent assay

The activities of other reductases which are similar to Trx or TrxR was determined in

the presence of PbTx-2. As mentioned above, Grx and GPx are both related to the

GSH reduction system to protect cells from oxidative damage by thiol-disulfide

exchange. Thus, it is anticipated that PbTx-2 may affect their activity as well. The

activity of Grx and GPx in the presence and absence of PbTx-2 was examined using

commercially available assay kits.

In Grx assay, the Grx activity was measured by a coupled reaction of GSH /GSSH

system. NADPH was added as electron donor to reduce GSSG to GSH via GR. The

oxidized Grx was reduced by GSH. In this kit, an eosin- modified GSH was used as

its substrate (Figure 44). An increase in fluorescence was recorded at 545 nm

emission and 520 nm excitation over 120 mins. The rate of reaction was treated as

activity of Grx.

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Figure 44 The principle of sensitive fluorescent assay for activity of Grx

In GPx assay, the GPx activity was measured indirectly by a coupled reaction with

GR. First, the reduction of hydroperoxide by GPx produced the oxidized GSSG.

Second, the GSSG is reduced by GR with NADPH. The oxidation of NADPH to

NADP+ can be monitored by a decrease in absorbance at 340 nm (Figure 45). Final,

GPx activity is directly proportional to the rate of decrease in the A340.

Figure 45 The reaction in sensitive fluorescent assay for GPx system No inhibition was observed for either enzyme in the presence of PbTx-2 (up to 56 µM

for Grx and 30 µM for GPx) (Figure 46 and Figure47). Because these kits contain GR

to provide a supply of reduced glutathione, we can conclude that PbTx-2 also does not

inhibit GR. Therefore, none of these oxidoreductases (Gx, GR or GPx) were inhibited

by PbTx-2 demonstrating the selectivity of PbTx-2 for TrxR.

Figure 46 The effect of Grx activity by PbTx-2. Reaction mixture containing 1 mM GSH, 54 µM NADPH and 0.1 µM GR was incubated with PbTx-2 (10 µl of the following standards: 0.95, 0.63, 0.32 and 0.0 mM in methanol) for 30 min. Human Grx 1 (16 µl, 30 nM), PBS (102

78

µl) and eosin labeled GSH (32 µl) were mixed with mixture. The final concentrations of PbTx-2 were, 0 µM, 11 µM, 22 µM, 33 µM

Figure 47 The effect of GPx activity by PbTx-2 PbTx-2 (15 µl of 2.2 mM, 1.3 mM and 0 mM in methanol) were each combined with the manufacturer provided assay buffer, GPx and GSH solutions to give PbTx-2 concentrations of 78 µM, 47 µM and 0 µM. Controls without GPx were treated as background. The final concentrations of PbTx-2 were 50 µM, 30 µM and 0 µM

3.4 Conclusion

Studying Trx inhibitors is complicated by the fact that the reaction between Trx and

it’s substrates is stoichiometric. This necessitates the amplification of the Trx signal.

Amplification can be performed at the back end of the Trx reaction as in the modified

papain assay. Alternatively, the Trx signal can be amplified by continuous reduction

of oxidized Trx with NADPH, catalyzed by TrxR. In either case, a two enzyme

system is used. The results from the modified papain assay suggest that brevetoxin

may be an inhibitor of Trx. However, the interpretation of the results of this assay are

complicated by the observation that brevetoxins are inhibitors of papain. The

TrxR/Trx activity assay demonstrates that PbTx-2 but not PbTx-3 is an inhibitor of

the mammalian TrxR/Trx system with an IC50 of 25 µM. However, the mechanism

and target of the inhibition is still unknown. The lack of inhibition of the TrxR/Trx

system by PbTx-3 suggests that the α,β-unsaturated aldehyde functionality of PbTx-2

is key to the inhibition of the Trx/TrxR system. The Ellman’s assay for TrxR activity

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shows that both PbTx-2 and to a lesser extent, PbTx-3 enhance the activity of

mammalian TrxR. The enhancement of DTNB reduction by TrxR observed in the

presence of PbTx-2 was unexpected. According to previous reports, small molecules

such as DTNB can be reduced by the N-terminal reaction center of TrxR when the C-

terminal center is either alkylated with electrophiles or absent as in the case of

truncated TrxR (Eckenroth, Lacey et al. 2007, Cheng, Sandalova et al. 2009, Lothrop,

Ruggles et al. 2009) It can be assumed that the modification of TrxR by PbTx-2

occurs at the C-terminal redox center. The observation that truncated TrxR which

lacks the C-terminal redox center, is unaffected by PbTx-2 also supports the

hypothesis that PbTx-2 affects mammalian TrxR at the C-terminal redox center.

Furthermore, the rate of DTNB reduction was not affected by pre-incubation of

reduced TrxR with PbTx-2 or addition of PbTx-2 and DTNB to pre-reduced TrxR. It

indicates that the alkylation of the C-terminal redox residue(s) of TrxR by PbTx-2

occurs rapidly relative to the reduction of DTNB. Crystal structures of TrxR with and

without Trx have shown that the C-terminal (Cys496/Sec497) residues of TrxR are

located at the end of a mobile arm. Prior to docking with Trx, the mobile arm is in

what is known as the “reduced waiting position” and the C-terminal residues are

buried in the interior of the enzyme. Upon docking with Trx, the mobile arm swings

out of this position to the surface of the enzyme (Fritz-Wolf, Kehr et al. 2011). It is

possible that alkylation with the large brevetoxin molecule also causes the mobile are

to move away from the N-terminal disulfide making it more accessible by DTNB,

bypassing the slow reduction of the C-terminal selenosulfide resulting in a rate

enhancement.

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Based on the adduct formed between TrxR and selenocysteine, it seems most likely

that modification of TrxR takes place at the Sec residue as selenols are better

nucleophiles than thiols: selenols have a lower pKa than thiols and would be

deprotonated to the selenide under physiological conditions. While our MS

experiments using TrxR and PbTx-2 did not provide direct evidence for Sec

alkylation, the conspicuous absence of the IAC modified C-terminal peptide,

SGGDILQSGCUG, which was identified in the control sample, suggests modification

at this site. Alkylation of this peptide by PbTx-2 would prevent IAC alkylation, and

the absence of this peptide suggests that the PbTx-2 modified peptide may be resistant

to trypsin digestion or may not ionize efficiently. Because the MS analysis of PbTx-2

treated TrxR failed to provide direct evidence of alkylation of Sec, the selenol

selective Sel-green probe was prepared to monitor for the presence of the reduced

selenol of TrxR in the presence and absence of PbTx-2. As anticipated, in the absence

of PbTx-2 the release of an equivalent of fluorescent coumarin reporter was observed

upon the addition of Sel-green to reduced TrxR. However, in the presence of PbTx-2

the observed fluorescence was significant lower which indicates that Sec had been

alkylated with PbTx-2.

3.5 Materials and Methods


The materials for the modified papain assay Papain, methyl methanethiosulfonate,

N-benzoyl-L-arginine-p-nitroanilide, immobilized TCEP (Tris [2-carboxyethyl]

phosphine hydrochloride), cystamine and thioredoxin 1 recombinant protein were

purchased from Thermo Scientific Co.. Brevetoxins (PbTx-2 and -3) were isolated

from K. brevis cultures according to published methods (Baden, Mende et al. 1981).

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UV/Visible measurements were performed in 96 or 384 well microplates using a

Synergy® 2 (Biotek Instrument, Inc.)

Preparation of papain-S-SCH3 (~1.2 mg / ml) Papain stock solution (1.5 ml, 26.4

mg / ml) was added to a solution of cysteine (0.6 mM, 28.5 ml of 20 mM sodium

phosphate – 1 mM EDTA buffer). The solution was kept at room temperature for 30

min. The methyl methanethiosulfonate (117 µmol) was added in mixture. The

reaction mixture was kept in ice for 4 hr. The mixture was dialyzed (Mr cutoff 6000-

8000) against 5 mM sodium acetate buffer, containing 50 mM NaCl, pH 4.7 at 4oC 12

hr.

The method for the modified papain assay The thioredoxin solution (1 mg / ml, 1

mM Tris – HCl pH.7) was incubated with immobilized TCEP for 1 hr. The reduced

thioredoxin solution (90 µl,1 µg / µl) was reacted with 20 µg brevetoxin (1 µg / µl,

PbTx-2 or PbTx-3) for 1 hr in one clean test tube. The solution of papain-S-SCH3

was diluted with an equal volume of 40 mM sodium phosphate buffer. The resulting

solution is ~0.6 mg / ml papain-S-SCH3 at pH 7.5, containing 1 mM EDTA. Papain-

S-SCH3 solution (0.5 ml), substrate (0.7 ml) and cystamine (15 µl, 26.4 mg / ml) were

added in the test tube with mixture. A tube containing papain-S-SCH3, without any

thiol, serves as the control. The mixture is carried out in triplicate in plate. Each well

was added 300 µl mixture. Record the absorbance at 410 nm every 60 min up to 15 hr.

3.5.2 Papain assay

The materials for the papain assay Papain, N-benzoyl-L-arginine-p-nitroanilide

and TCEP were purchased from Thermo Scientific Co. Brevetoxins (PbTx-2 and -3)

were isolated from K. brevis cultures according to published methods (Baden, Mende

et al. 1981). UV/Visible measurements were performed in 96 or 384 well microplates

using a Synergy® 2 (Biotek Instrument, Inc.)

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The method for the papain assay The solution of papain (304 µl, 26.4 mg / ml ), N-

benzoyl-L-arginine-p-nitroanilide (0.7 ml) were added in the test tube with mixture.

PbTx-2 and PbTx-3 (20 µl, 1 mg / ml) was added to the tube respectively. Using the

Tris buffer filled the finial volume to 625 µl. A tube without any brevetoxin, served as

the control. The mixture is carried out in triplicate in plate. Each well was added 300

µl mixture. Record the absorbance at 410 nm every 60 min up to 15 hr.

3.5.3 Fluorescent assay for Trx/TrxR system

The materials for the fluorescent assay for Trx/TrxR system: Unless otherwise

noted, reagents were purchased from Sigma-Aldrich or Acros chemical companies

and used without further purification. Brevetoxins (PbTx-2 and -3) were isolated from

K. brevis cultures according to published methods (Baden, Mende et al. 1981).

Enzyme assay kit was purchased from Cayman Chemical. Assay kit was used

according to the manufacturer’s instructions with noted exceptions. Fluorescence

measurements were performed in 96 or 384 well microplates using an Infinite®

M1000 PRO (Tecan Group Ltd.) microplate readers.

The methods for the fluorescent assay for Trx/TrxR system. Experiments were

performed in 96-well plates with 270 µl of assay buffer (50 mM Tris-HCl, 1 mM

EDTA, 0.14 mg / ml BSA, pH 7.5) containing 0.13 µM rat TrxR, 0.35 mM NADPH

and 0.026 µM Trx. After incubation for 30 min at 37 0C to reduce the TrxR, stock

solutions of PbTx-2 or PbTx-3 (11 µl each of 1.1, 0.96, 0.64, 0.35 and 0 mM in

methanol) were added to give final toxin concentrations of 35 µM, 30 µM, 20 µM, 11

µM, 0 µM. This mixture was incubated at 37 0C for 1 h. Fluorescent substrate (70 µl,

83

0.4 mg / ml, FiTC labeled bovine insulin) was added to each aliquot (final

concentration of 0.016 mg / ml). Samples were analyzed in triplicate by recording

fluorescence at 545 nm (λex = 520 nm) every 10 min for 3 h.


The materials for the DTNB assay: Unless otherwise noted, reagents were

purchased from Sigma-Aldrich or Acros chemical companies and used without

further purification. Brevetoxins (PbTx-2 and -3) were isolated from K. brevis

cultures according to published methods (Baden, Mende et al. 1981). Truncated

human TrxR (Novus Biologicals) was purchased from Fisher Scientific. Rat liver

TrxR was purchased from Cayman Chemical. DTNB was purchased from Thermo

Scientific Co.. NADPH was purchased from Molecular Toxicology, Inc. UV/Visible

measurements were performed in 96 or 384 well microplates using a Synergy® 2

(Biotek Instrument, Inc.)

The method for the DTNB assay for TrxR or truncated TrxR. Method A (pre-

incubation with PbTx-2): Experiments were performed in 96-well plates with 160 µl

of PBS containing 0.1 mM NADPH. 22 µM PbTx-2 and 0.56 µM rat TrxR or 0.68

µM truncated human TrxR. After incubation for 30 min at 37 0C to reduce the TrxR,

40 µl of a DTNB solution (Ellman’s reagent: 5,5’-dithio-bis-(2-nitrobenzoic acid) (10

mM in 0.1 M sodium phosphate and 1 mM EDTA, pH 8.0) was added for final

concentrations of 18 µM PbTx-2 and 2 µM DTNB. The absorbance at 410 nm was

measured every 5 min for up to 60 min. Method B (no pre-incubation with PbTx-2):

The assay was performed as described above except that PbTx-2 and DTNB solutions

were mixed and added simultaneously after NADPH reduction of TrxR.

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3.5.5 The preparation of selenocysteine -PbTx-2 adduct

The materials for making selenocysteine-PbTx-2 adduct PbTx-2 isolated from K.

brevis cultures according to published methods (Baden, Mende et al. 1981). Seleno-L-

cysteine was borrowed from Dr. Lees lab. Immobilized TCEP was purchased from

Thermo Scientific Co.. The formation was detected from a Bruker Solarix 7.0 T using

ultrahigh resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

(FT-ICR).

Preparation and analysis of selenocysteine-PbTx-2 adduct seleno-L-cysteine(75

µL, 1 mg /mL) was incubated with an aqueous suspension of immobilized TCEP

((tris(2-carboxyethyl) phosphine, 30 µL, >8 µmol/mL, immobilized onto 4%

crosslinked beaded agarose) for 1 h to reduce the diselenide. The reaction vial was

flushed with nitrogen and centrifuged for 10 min at 14,000 x g. The supernatant was

transferred via syringe to a solution of PbTx-2 (200 µL, 1 µg/µL in methanol) and

stirred under nitrogen for 24 h. Accurate mass spectra was acquired using a Bruker

Solarix 7.0 T using ultrahigh resolution Fourier Transform Ion Cyclotron Resonance

Mass Spectrometer (FT-ICR) confirmed the formation of an oxidized (+15.999 amu)

selenocysteine-PbTx-2 adduct of formula C53H77NO17Se. HRMS calc’d for [M+H+]:

1080.44355. Found: 1080.44356 (Δ = -0.56 ppm). HRMS calc’d for [M+Na+]:

1102.42545. Found: 1102.42550 (Δ = -0.12 ppm).

3.5.6 The preparation of TrxR-PbTx-2 adduct.

The materials for making TrxR-PbTx-2 adduct Rat liver TrxR was purchased from

Cayman Chemical. PbTx-2 isolated from K. brevis cultures according to published

85

methods (Baden, Mende et al. 1981). NADPH was purchased from Molecular

Toxicology, Inc..

Preparation and analysis of TrxR-PbTx-2 adduct. Rat TrxR (6 µM) and NADPH

(0.16 mM) were incubated for 30 min in 44 mM Tris base, 0.9 mM EDTA. 0.4 M

NaCl, 0.1 % glycerol, pH 7. PbTx-2 (5.6 mM in Me2SO) was added to give a final

PbTx-2 concentration of 0.24 mM and the reaction was incubated for 12 h at room

temperature. The sample was concentrated 5 fold and analyzed by SDS PAGE (12%

acrylamide, 10% SDS). The band was excised and stored at -80 °C. Gel digestion and

LC-Electrospray Ionization MS/MS (LC-ESI MS/MS) analysis was performed by the

Proteomics and Mass Spectrometry Facility at the UMass Medical School as

previously described.

3.5.7 Reaction of Sel-green probe with TrxR

The materials for reaction of sel-green with TrxR Sel-Green was synthesized by

Freddy Rodriguez. Rat liver TrxR was purchased from Cayman Chemical. PbTx-2

isolated from K. brevis cultures according to published methods (Baden, Mende et al.

1981). NADPH was purchased from Molecular Toxicology, Inc. Seleno-L-cysteine

was borrowed from Dr. Lees lab. Fluorescence measurements were performed in 96

or 384 well microplates using a an Infinite® M1000 PRO (Tecan Group Ltd.)

microplate readers.

Reaction of Sel-green probe with TrxR. The selenol selective probe, Sel-green, was

synthesized according to published methods (Zhang, Ge et al. 2015). Rat TrxR (6

86

µM) and NADPH (0.1 mM) were incubated for 30 min in 45 mM Tris base, 0.9 mM

EDTA. 0.5 M NaCl, 0.1 % glycerol, pH 7. PbTx-2 (1.1 mM in methanol) was added

for a final toxin concentration of 48 µM. Samples were prepared in triplicate. A

control sample was prepared by the addition of an equal volume of methanol without

PbTx-2. A blank sample was prepared without TrxR. The solutions were incubated

for 1 h. Sel-green probe (9 µl, 481 µM in 20% acetone in PBS) was added to each

reaction mixture (82 µl). After 55 min, fluorescence at 502 (λex = 370 nm) was

measured. Calibration standards were prepared as follows: A stock solution of seleno-

L-cysteine (1.0 ml, 27 µM, in PBS) was reduced using immobilized TCEP (1.0 ml, >

8 µmol/ml) for 1 h. After centrifugation (14000 x g, 2 min), the supernatant was

transferred to a clean tube to provide a stock solution of L-selenocysteine (54 µM).

Aliquots of the stock solution of L-selenocysteine was diluted with Sel-green probe in

PBS for final concentrations of L-selenocysteine of 0 µM, 3 µM, 6 µM, 12 µM, 25

µM and 43 µM Sel-green probe, incubated for 55 min, and fluorescence was

measured at 502 nm (λex = 370 nm). Calibration standards were prepared in

triplicate. For this experiment, the concentration of FAD (flavin adenine dinucleotide)

was calculated to be 5.3 µM by measuring the absorbance of at 450 nm and using an

extinction coefficient ε450 = 11,300 M–1cm–1 for FAD (Zhong and Holmgren 2000).

3.5.8 Fluorescent assay for Grx and GPx

The materials for the activity assay for Grx Fluorescent Grx kit was purchase from

Cayman Chemical. PbTx-2 isolated from K. brevis cultures according to published

methods (Baden, Mende et al. 1981). Fluorescence measurements were performed in

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96 or 384 well microplates using an Infinite® M1000 PRO (Tecan Group Ltd.)

microplate readers.

The method for the activity assay for Grx. Experiments were performed in 96-well

plates with 160 µl of a reaction mixture containing 0.17 M potassium phosphate, 1.7

mM EDTA, 1 mM GSH, 0.1 mg / ml BSA, 54 µM NADPH, 0.1 µM GR, pH 7.5

which was prepared according to the manufacturer’s instructions. To aliquots (160 µl)

of the reaction mixture was added PbTx-2 (10 µl of the following standards: 0.95,

0.63, 0.32 and 0.0 mM in methanol), 16 µl of human Grx 1 (30 nM in 0.83 M

potassium phosphate and 8.3 mM EDTA, pH 7.5, final concentration 1.5 nM), 102 µl

of PBS and 32 µl of eosin labeled GSH bound to BSA (0.2 mM in distilled water).

Samples were analyzed in triplicate by recording fluorescence at 545 nm (λex = 520

nm) every 10 min for up to 2 h. The final concentrations of PbTx-2 were, 0 µM, 11

µM, 22 µM, 33 µM.

The materials for the activity assay for GPx Fluorescent GPx kit was purchase

from Cayman Chemical. PbTx-2 isolated from K. brevis cultures according to

published methods (Baden, Mende et al. 1981)Fluorescence measurements were

performed in 96 or 384 well microplates using an Infinite® M1000 PRO (Tecan

Group Ltd.) microplate readers

The method for the activity assay for GPx The assay was carried out according to

the manufacturer’s instructions except that stock solutions of PbTx-2 (15 µl of 2.2

mM, 1.3 mM and 0 mM in methanol) were each combined with the manufacturer

provided assay buffer, GPx and GSH solutions to give PbTx-2 concentrations of 78

µM, 47 µM and 0 µM. This mixture was incubated for 1 h at room temperature, after

88

which the assay was completed according to the manufacturer’s instructions. The

absorbance was read once every minute at 340 nm for 10 min. Samples were analyzed

in triplicate. Controls without GPx were treated as background. The final

concentrations of PbTx-2 were 50 µM, 30 µM and 0 µM.

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Chapter 4 Comparison of biochemical parameters in high toxin and low toxin

producing strains of K. brevis.

4.1 Objective

Compare and contrast various metabolic and biochemical parameters in two strains of

K. brevis which have a tenfold difference in toxin content

4.2 Introduction

In chapter 2 and 3, by using fluorescent and photoaffinity derivatives of brevetoxin it

was concluded that brevetoxin localizes to the chloroplast of K. brevis where it

interacts with two cholorplast proteins: LHCII and Trx (Cassell, Chen et al. 2015). It

was also shown that brevetoxin inhibits the Trx/TrxR system within an IC50 of 25 µM.

In addition to inhibition of the Trx/TrxR system, brevetoxin affects mammalian TrxR

leading to the enhancement of the activity of this enzyme. The evidence indicates that

brevetoxin affects mammalian TrxR at the C-terminal selenocysteine active site

residue. These discoveries lead us back to our original question: what is the

biochemical role of brevetoxin in K. brevis? Or more specifically: what are the

implications of TrxR inhibition by brevetoxin for K. brevis? In fact, LHC II, Trx and

TrxR are all common antioxidants related to reactive oxygen species (ROS) in cells.

When the excess ROS accumulate in cells, the state is called oxidative stress. Various

biomolecules such as proteins (Stadtman 1992, Nordberg and Arnér 2001), lipids,

lipoprotein S (Yla‐Herttuala 1999), and DNA (Marnett 2000, Nordberg and Arnér

2001), are threatened by the reactive oxygen intermediates. Oxidative stress is

proposed to be involved in DNA damage in the mitochondria (Cadenas and Davies

2000, Finkel and Holbrook 2000). However, a small concentration of ROS is required

for normal cell function as well. It has been demonstrated that ROS are involved in

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intracellular signaling and redox regulation. The term ROS includes a number of

chemically reactive molecules which are derived from oxygen (Galter, Mihm et al.

1994, Halliwell 1996, Fridovich 1999, Betteridge 2000) (Table 9). Some are

extremely reactive like hydroxyl radical, in contrast some are less reactive, such as

superoxide anion (O2-) and hydrogen peroxide (Nordberg and Arnér 2001) Table 9

also shows the main sources of production and the enzymatic defense system for

various ROS.

Table 9 The major ROS molecules and their metabolism

ROS molecule Enzymatic defense systems Products Superoxide (O2

-) Superoxide dismutase (SOD), Superoxide reductase

H2O2+O2 H2O2

Hydrogen peroxide (H2O2)

Glutathione peroxidase, Catalases,

Peroxiredoxins (Prx)

H2O+GSSG H2O+O2

H2O

Hydroxyl radical (.OH)

Nitric oxide (NO) Glutathione/TrxR GSNO

Superoxide anion (O2-) is formed from molecular oxygen. Although it is a free

radical, it is not highly reactive. Because it cannot penetrate lipid membranes, which

means superoxide anion can only remain where it is produced (Nordberg and Arnér

2001). Usually, superoxide anion is formed in the electron-rich aerobic environment

such as the inner mitochondrial membrane with the respiratory chain (Derick,

Williams et al. 2001). Lipoxygenase and cyclooxygenase are also generated

superoxide (Kontos, Wei et al. 1985, Mattei, Virdis et al. 1996). In addition to

superoxide, hydrogen peroxide has similar intracellular effects (Sundaresan, Yu et al.

1995, Finkel 1998, Kamata and Hirata 1999, Rhee 1999, Patel, Moellering et al.

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2000). Hydrogen peroxide is a highly important ROS molecule because H2O2 is able

to penetrate biological membranes. Many antioxidant enzyme systems, such as

catalase, glutathione peroxidase and peroxiredoxins, are able to remove H2O2 (Chae,

Kang et al. 1999, Chae, Kim et al. 1999, MatÉs, Pérez-Gómez et al. 1999). Hydroxyl

radical (.OH) is the most reactive ROS molecule, causing more damage to biological

systems than other ROS. Usually .OH is obtained from H2O2 (Neyens and Baeyens

2003). Last but not least is nitric oxide (NO), a well-known as a signaling molecule.

Additionally, NO has been identified as an inhibitor of lipid peroxidation in cell

membranes (Hogg and Kalyanaraman 1998, Rubbo, Radi et al. 2000). NO is able to

regulate gene transcription and expression as well (Bogdan 2001).

Due to the high reactivity of ROS, it causes damage to numerous biomolecules. For

example, ROS is treated as a mutagen. ROS causes a number of alterations such as

DNA cleavage, DNA protein cross-linking and oxidation of purines when the cells are

exposed to oxidative stress. It is determined that ROS are related to DNA mutation,

cancer and apoptosis (Halliwell, Zentella et al. 1997, Kamata and Hirata 1999). Lipids

are also an excellent target for ROS because of the multiple double bonds in

polyunsaturated fatty acids (Steinberg 1997, Kamata and Hirata 1999). Lipid

peroxidation is essential for the generation of atherosclerotic plaques (Halliwell 1993,

Frei 1999). The incidences of cardiovascular disease are increasing in western

countries. The most important prevention and medical treatments are to decrease lipid

peroxidation, which is associated with excessive amounts of ROS. Furthermore, ROS

has been shown to react with several amino acids in vitro, resulting in modification or

inhibition of the activity of enzymes (Stadtman and Berlett 1997, Butterfield, Koppal

et al. 1998).

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ROS are affected by antioxidants such as the Trx and GSH systems (Lowther, Brot et

al. 2000, Nordberg and Arnér 2001). LHCII also affects ROS production. In fact,

plants and algae love light but also hate light. Oxygenic photoautotrophic organisms

require light for life. However, when the input of excess high light exceeds the

saturation limit of the photosynthetic apparatus this leads to an increase the

production of ROS. Chloroplasts have evolved a number of mechanisms to mitigate

the damaging effects of ROS, which include both the scavenging of ROS and the

suppression of ROS production through NPQ (Figure 48 A). When a Chl absorbs

light energy, it is excited from ground state to singlet molecule (1Chl*). The singlet

1Chl* can be re-emitted as Chl fluorescence that is used as photochemistry. Also, it

can be de-excited by NPQ. However, singlet 1Chl* is able to turn to triplet state (3Chl*)

which can transfer energy to ground state O2 to form singlet oxygen (1O2*). Singlet

oxygen (1O2*) is an extremely damaging ROS. In normal light, the energy is

transferred to photochemistry. The yield of Chl fluorescence is generally low (Krause

and Weis 1991). However, under excess light, the increase of 1Chl* leads to yield high

level of 3Chl* which generates more 1O2*. 1O2

* causes oxcide damage to the cells

(Figure48 B). However, NPQ can either dissipate the 1Chl* as heat or quench Chl

fluorescence to prevent cells from oxygen damage (Müller, Li et al. 2001). Thus,

NPQ is one of mechanisms to suppress to ROS production. If the cells cannot perform

NPQ to dissipate light energy, more ROS will be produced. As mentioned above,

LHCII is an integral part of the photosynthetic apparatus where it harvests and

distributes light energy using four LHCII bound carotenoid molecules. It is also

central to the process of NPQ or the dissipation of light energy as heat under

conditions of high light.

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Figure 48 The common pathways to produce ROS. First, high light energy is transfered to photochemistry. (A) The common pathway to distribute high light energy in leaves. Once the photochemistry reaches saturation, the excess energy can either be dissipated to heat by NPQ or switch to chlorophyII fluorescence. (B) The possible way to release excited Chl. When Chl absorbs light it is excited from the ground state to the singlet excited state. The singlet excited form, 1Chl* can go back to the ground state by three ways: emitting light as fluorescence, turning to heat and being used in photosynthetic reactions (Müller, Li et al. 2001) In addition to LHC II, in photosynthetic organisms, the Trx/TrxR system is located

within the chloroplast lumen and numerous thioredoxin-dependent regulatory

networks have been identified within the chloroplasts of photosynthetic organisms

(Nikkanen and Rintamäki 2014), including VDE activity (Hall, Mata‐Cabana et al.

2010). In plants, there are highly versatile Trx systems which include two different

TrxRs: ferredoxin thioredoxin reductase (FTR) and NADPH dependent thioredoxin

reductase (NTRC). These TrxRs are localized to plant chloroplast. They contribute to

the regulation of proteins in chloroplast by Trx system. Additionally, it is believed

that the two reductases are selective for different Trx targets. FTR is active only in the

light and is believed to be responsible for regulation of light reactions. While NTRC

enzyme is unique for oxygenic photosynthetic organisms (Nikkanen and Rintamäki

2014).

In mammal, Trxs are also critical to the scavenging of excess ROS by reduction of 2-

cysteine peroxiredoxins (2-Cys Prxs), enzymes whose sole function is the

94

detoxification of peroxides (König, Baier et al. 2002) It is reported that the expression

of Trx and TrxR are induced by oxidative stress in adult T cells (Nakamura, Matsuda

et al. 1994, Taniguchi, Taniguchi-Ueda et al. 1996). When the cells are exposed to

oxidative stress, Trx/TrxR system is the sophisticated antioxidant regulatory system to

maintain the balance of ROS. In Figure49, the major physiological functions of the

mammalian thioredoxin system are shown. In particular, mammalian TrxR, which

reduces more substrates than mammalian Trx, reduces not only the disulfide in

oxidized Trx, but also different protein disulfides, PDI, low molecular weight

disulfides such as DTNB, and nondisulfide compounds, such as hydrogen peroxides,

and nitroblue tetrazolium (NBT)(Arnér, Zhong et al. 1999, Nordberg and Arnér

2001). Mammalian TrxR reduces some selenium-containing enzymes and compounds

either directly or by way of Trx (Kumar, Bjornstedt et al. 1992). For examples,

selenite and selenocysteineand sec-containing GPx are reduced by mammalian TrxR

as well. The active site of GPx is regenerated directly by TrxR (Nordberg and Arnér

2001). It has been determined that sulfur and selenium compounds are able to reduce

the ROS generation through their antioxidant properties. For example, patients with

pulmonary tuberculosis show reduced oxidative stress after taking selenium

supplementation (Seyedrezazadeh, Ostadrahimi et al. 2008). Commonly, sulfur and

selenium compounds protect against disease through ROS radical scavenging and

enzymatic decomposition of oxygen metabolites (Mugesh and Singh 2000, Valko,

Rhodes et al. 2006).

95

Figure 49 Major physiological function of the mammalian thioredoxin system

Furthermore, there are other cellular antioxidant enzyme systems protecting cells and

organisms from excessive ROS. Superoxide dismutase (SOD) catalyzes the reaction

of O2*- (Yost and Fridovich 1973) to produce molecular oxygen and hydrogen

peroxide. Catalases are mainly heme-containg enzymes. Catalases catalyze the

conversion of hydrogen peroxide to water and molecular oxygen (Aebi 1984). All

GPx catalyze the reduction of H2O2 through the assistance of the GSH system. The

catalytic mechanism was proposed in Chapter 3. Last but not least, the GSH system

is the most abundant intracellular thiol-based antioxidant. GSH can be present in

millimolar concentration in living cells (Armstrong 1997, Van Bladeren 2000). GSH

serves as a sulfhydryl buffer to protect against oxidative damage in cells. The

oxidized GSSG can also be reduced by Trx system(Kanzok, Schirmer et al. 2000).

Both LHCII and Trx regulate the oxidative state of the cell and redox signaling. Both

affect the amount of ROS production. In addition to these brevetoxin targeted

proteins, the GSH system and many antioxidants are involved in preventing oxidative

stress in cells. The finding that brevetoxin binds to LHCII and Trx raises an important

question: Does brevetoxin influence the ROS level and the antioxidant content in K.

96

brevis through its interaction with to LHC II and the TrxR/Trx system? In the work

presented here, two different strains of K. brevis which have a tenfold difference in

toxin content (Sun, Leeson et al. 2016), had been evaluated and compared for redox

status.

In 2015, we determined the rate of production of ROS and the NPQ performance in

high-toxin producing and low-toxin producing K. brevis strains. It was recorded by

using a cell permeable ROS reactive indicator dye, known as carboxy-H2DCFDA

(Figure 50).

Figure 50 The structure of carboxy-H2DCFDA

Live K. brevis cells were mixed with carboxy-H2DCFDA in 96-wells microplates.

After hydrolysis of the acetate groups by esterases, the dye is oxidized to a fluorescent

product. After normalizing to chlorophyll fluorescence it was found that the low toxin

culture produced ROS at twice the rate of the high toxin producing culture (Figure 51)

(Cassell, Chen et al. 2015).

Figure 51 The ROS production in two strains of K. brevis. ROS production was normalized to chlorophyll fluorescence. Low toxic strain produces more ROS than high toxic strain

0

50

100

150

200

0 2 4 6 8

RO

S (n

M) /

Chl

l)

Time (hr)

ROS production in K. brevis Low Toxin High Toxin

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NPQ of two strains of K. brevis was determined during log phase growth by pulsed

amplitude modulation (PAM) fluorimetry at four excitation wavelengths (Cassell,

Chen et al. 2015). Under the same culture conditions, the toxic culture reached an

algal biomass of about 1590 mg Chl / L, versus 240 µg Chl / L for the less toxic

culture, after 20 days of growth. Both algal cultures exhibited the same fluorescence

yield subsequent to the pulses of four different excitation wavelengths (470, 520, 645,

and 665 nm). The NPQ was calculated for each excitation wavelength by measuring

the saturated fluorescence at 3600 mmol photons/ m2 . s of the 25 min dark and actinic

light adapted (363 mmol photons / m2 . s) algal cultures. NPQ for the four excitation

wavelengths were determined at increasing levels of 1 min actinic light adaptation,

ranging from 164 to 2064 mmol photons / m2 . s. NPQ increased with increasing

actinic light intensities, but this increase was much lower for the less toxic culture and

overall much less than for the toxic culture (Figure 52). To our surprise, a slightly

negative NPQ (-0.05 to - 0.01) was observed for the less toxic culture at actinic light

intensities 64–764 mmol photons / m2 . s, whereas all NPQ values were positive for

the toxic culture.

Figure 52 NPQs for the low and high toxic algal cultures to the pulses of four different excitation wavelengths (470, 520, 645, and 665 nm). It was calculated for each sample after 1 min of adaptation to increasing levels of actinic lights. Both cultures exhibited an increase in NPQ with actinic light, but a sharper increase in

-5% 5%

15% 25% 35% 45% 55% 65%

0 400 800 1200 1600 2000

NPQ

Actinic light (µmol.photons / m2. s)

NPQ performance in K. brevis

98

magnitude of the NPQs were found for the toxic culture. (Light toxic strain in 470 nm, 520 nm, 645 nm, 665 nm, high toxic strain in 470 nm, 520 nm, 645 nm, 665 nm) The NPQ and ROS experiments revealed that the high toxic strain can perform NPQ

to prevent cells from damage by ROS. In fact, the low-toxic strain produces ROS at

about twice the rate of the high toxic strain, and does not perform NPQ as efficiently

as the high toxic strain. It is unclear if the lack of toxin causes the NPQ deficiency or,

if the NPQ deficiency results in lower toxin production.

4.3 Results and discussion

Initially we had assumed that K.brevis TrxR would be more like that of a plant. Plant

TrxR is more similar to prokaryotic TrxR which does not have the Cys-Sec. However,

it is reported a “mammalian like” TrxR was found in Emeliania huxleii which has the

C-terminal selenosulfide by researching the gene sequence (Araie, Suzuki et al. 2008).

Thus, it is very likely that K. brevis does has “mammalian like TrxR. Using the

sequence from Emeliania huxleii as a query in a BLAST search of the K. brevis

transcriptome library it is revealed that two “mammalian like” TrxR sequence in

which the C-terminal selenosulfide is present (Darcie, Alan et al. 2014).

UGA codon is normally a termination codon, whereas UGA codon is able to code Sec

in a special way. A selenocysteine insertion sequence (SECIS) presents in the mRNA,

which is responsible to make UGA codon to encode Sec (U). SECIS is defined as a

nucleotide sequence which typically located following the UGA codon. In Archaea

and eukaryotes, the SECIS element is located in the 3’ untranslated region of the

mRNA. The SECIS element was found in both DNA sequence of TrxR in K. brevis.

In addition, a selenocysteine residue was discovered in one amino acids sequence of

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TrxR in K. brevis, which was aligned with human TrxR and “mammalian like ” TrxR

sequence in Emeliania huxleii (Table 10)

Table 10 The alignments of TrxR in K. brevis, Homo sapiens and Emiliania huxleyi

4.3.1 The antioxidants in two strains of K. brevis

Antioxidants play an important role in protecting cells from oxidative stress. GSH,

ascorbic acid, Trx, carotenoids and vitamin E are central to the antioxidant defense in

most of species. Thus, it is suspected antioxidant capacity may not be same between

high toxic and low toxic strain. Examining the antioxidant capacity and ascorbic acid

content may contribute to understanding the role of brevetoxin in K. brevis.

Here, the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay was

used to detect all antioxidants such as lipophilic carotenoids, GSH, Trx and ascorbic

100

acid (Figure53). The ABTS assay measures the decolorization (reduction) of the

ABTS radical cation which has a long wavelengthabsorption (Re, Pellegrini et al.

1999).

Figure 53 The reaction in ABTS assay

Total antioxidant capacity of K. brevis homogenates was determined using ABTS

radical cation (ABTS.+) decolorization (Re, Pellegrini et al. 1999) and expressed as

trolox equivalents. Trolox is similar to vitamin E which is used in biological

applications to reduce oxidative damage. Trolox equivalent antioxidant capacity

(TEAC) is commonly used as a standard in the ABTS assay for measuring the

concentration of antioxidant in unknown sample. Here, the homogenate of two K.

brevis strains or trolox standard was used to decolorizw the ABTS!+ in the assay. The

concentration of total antioxidant was estimated based on the trolox concentration

standard curve. Furthermore, ascorbic acid concentration (as trolox equivalents) in K.

brevis homogenates was estimated by incubating with ascorbic acid oxidase (AAO).

AAO catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Thus, the

difference of absorbance in the presence and absence of AAO provides an estimate of

ascorbic acid concentration in the homogenate. Total antioxidant or ascorbic acid

content were normalized to protein concentration. It was shown that the high toxic

101

strain has a 1.2-fold antioxidant capacity than the low toxic strain on three of the four

days tested (Figure 54). In contrast, the low toxic strain consistently had higher levels

of ascorbic acid (Figure55). It showed that the low toxic strain has maximum 4.5-fold

of ascorbic acid at 19 growing days, when it compared to high toxic strain.

Figure 54 The antioxidant capacity in two strains of K. brevis. ABTS�+ was produced by oxidation of aqueous ABTS (1.3 mL, 7 mM) with ammonium persulfate (21.2 µl, 2.45 mM). The Either 50 µl K. brevis homogenate or trolox standards (0 µM, 25 µM, 50 µM, 100 µM, 200 µM) were added to the ABTS.+ solution (100 µl). The absorbance was read at 734 nM after incubation for 5 min

Figure 55 The total amount of ascorbic acid in two strains of K. brevis. Ascorbic acid concentration (as trolox equivalents) of K. brevis homogenate was estimated by incubating with of ascorbic acid oxidase (2 µl, 0.01 U/ µL) for 30 min prior to the assay and comparing absorbance to parallel samples without AAO

500.00

1000.00

1500.00

2000.00

12 Days 19 Days 20 Days 21 Days Trol

ox e

quiv

alen

t ant

ioxi

dant

ca

paci

ty (n

mol

/mg)

Antioxidant capacity in high toxic and low toxic strain

High toxic strain Low toxic strain

0.00

200.00

400.00

600.00

800.00

12 Days 19 Days 20 Days 21 Days Trol

ox e

quiv

alen

t am

ount

of

asco

rbic

aci

d (µ

mol

/ m

g)

Total amount of ascorbic acid in high toxic and low toxic strain


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4.3.2 Redox state of protein and non-protein thiols in low vs high toxin cultures

of K. brevis

Because brevetoxin is an inhibitor of the Trx/ TrxR system, it follows that the

presence of brevetoxin may impact the amount of free thiols in K. brevis. Therefore,

we examined the thiol content in high and low toxin producing strains of K. brevis.

Modified papain assay As mentioned in Chapter 3, this assay is more sensitive to

thiols than the Ellman’s assay as the signal is amplified by the enzymatic activity of

papain. Thus, the thiol content, normalized to protein concentration, was determined

first by using the modified papain assay in the high toxin and low toxin K. brevis

strains. Because the free thiol groups may be buried in proteins and not readily

accessible by modified papain, cystamine was added into the reaction to access not

only low MW thiols, but also free thiols in proteins. This experiment revealed a

higher thiol content is in the low toxic homogenate compared to toxic strain either

under light or dark conditions (Figure56). No matter under dark adapter or sun light, a

two-fold reduction of V0 was shown in the low toxic strain compared to high toxic

strains for the modified papain assay. Again, this assay is complicated by the fact that

brevetoxin is an inhibitor of papain. Thus, Ellman’s assay has to be used to detect the

thiol content in K. brevis homogenate.

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Figure 56 The detection of thiols in K. brevis homogenate by modified papain assay. The culture was either dark adapted or light adaptedfor 1 hr before harvesting cells. Papain-S-SCH3 solution (0.5 ml, 0.6 mg / ml), 0.7 ml substrate and 15 µl cystamine were added in one test tube which contained 200 µl supernatant. ( Dark adapted low toxic strain, Dark adapted high toxic strain, Light adapted low toxic strain, Light adapted high toxic strain)

Ellman’s assay The content of low molecular weight thiols in K. brevis homogenates

of both low and high toxic strains was also determined using the Ellman’s assay. This

reaction is stoichiometric, allowing for accurate quantification of free thiols.

Considering that the stage of the culture on a growth cycle may affect the thiol

content of cells, the free thiol content was monitored using DTNB assay during the

log phase of the growth cycle (Figure 57).

Figure 57 The concentration of cells over the lifetime of high toxin and low toxic strains

The thiols detected by the Ellman’s assay are believed to be composed principally of

reduced glutathione as cellular glutathione levels are typically in the mM range.

0

0.5

0 200 400

Abs

. / P

rote

in ( µg

/ µl

)

Time (min)

The detection of thiol in K. brevis homogenate by modified papain assay

A

B

104

However, other low molecular weight thiols and some higher molecular weight thiols,

which are accessible to DTNB may also be detected. Additionally, on selected days,

GR was added to parallel samples. GR will reduce any glutathione which is present as

the disulfide GSSG to the thiol GSH. In this way the total GSH can be quantified.

Also, the ratio of native GSH to total GSH can be calculated. At the end, the amount

of thiol can be obtained from the proportion of absorbance compared to the

concentration of cysteine standards. Then the amount of thiol was normalized to

protein concentration in the homogenate. It is clear that the low toxic strain has a

higher thiol concentration than high toxic strain, which is consistent with the previous

modified papain assay (Figure 58). From 17th to 19th days, low toxic strain produced

two folds of thiol content than high toxic strain. In 12, 21, 23 and 25 days, a 1.5-fold

of thiol content was produced in low toxic strain compared to high toxic strain.

However, after adding the GR prior to the assay and comparing absorbance to parallel,

it is seemed that two strains had similar amount of total GSH when the cells in the log

phase of growth (Figure 59 A). Finally, the ratio of reduced GSH to total GSH was

calculated based on the normalized results of before and after adding GR. It has been

shown that from 17th to 19th days low toxic strain has two-fold ratio of reduced GSH

to total GSH than high toxic strain (Figure 59 B). From 23 to 25 days, low toxic strain

has 1.5-fold ratio of reduced GSH to total GSH than high toxic strain.

0

200

12 days

17 days

19 days

21 days

23 days

25 days

Thi

ol /

prot

ein

( nm

ol /

mg)

The conc. of thiol in high toxic and low toxic strains

High toxin strain Low toxin strain

105

Figure 58 The concentration of thiol in high toxic and low toxic strains. K. brevis homogenates (300 µl, 0.3-0.5 mg / ml protein) was mixed with reaction buffer (700 µl, 0.1 M sodium phosphate, 1 mM EDTA, pH 8.0) and DTNB (20 µl, 4 mg / ml). Samples were quantitated against cysteine standards (0, 0.3, 0.6 and 1.2 mM) prepared in reaction buffer in place of homogenate by using the DTNB. The amount of thiol in homogenate was normalized to protein concentration in the homogenate.

Figure 59 The total glutathione in two strains of K. brevis and the ratio of native reduced glutathione to total glutathione in two strains of K. brevis. (A) K. brevis homogenates (275 µl, 0.3-0.5 mg / ml) was mixed with NADPH (100 µl, 1 mg / ml), glutathione reductase (3 µl, 10 µM) and Ellman’s reagent (25 µl, 4 mg / ml). Samples were quantitated against cysteine standards (0, 0.3, 0.6 and 1.2 mM) prepared in reaction buffer in place of homogenate by using the Ellman’s reagent. The amount of thiol in homogenate was normalized to protein concentration in the homogenate (B) the ratio of native GSH to total GSH was calculated based on the normalized results from before and after adding GR

4.3.3 The reaction of Sel-green probe in two K. brevis strains.

In previous experiments, the selenol selective probe was used to react with TrxR in

the presence or absence of PbTx-2. It was revealed that in the presence of PbTx-2 less

fluorescent reporter was released in the reaction of TrxR with sel-green when

compared to to the absence of PbTx-2. Here, the sel-green probe was used to react

with the homogenate of two different brevetoxin producing strains of K. brevis. After

incubation of homogenate for 1 hr, the fluorescence increase was monitored (ex/em =

370/502 nm). In the homogenate the thiol groups and selenol groups react with Sel-

green probe, resulted in release of the fluorescent reporter. If Sec is alkylated by

brevetoxin in homogenate, then the fluorescence reporter should not be released in the

presence of brevetoxin and fluorescence should not be observed. Fluorescence is

clearly reduced in the low toxin producing strains (Figure 60). It is highly likely that

0 1000 2000 3000 4000 5000 6000 7000

17 days 19 days 23 days 25 days

Tota

l glu

tath

ine

( nm

ol /

mg)

The conc. of native GSH in two K. brevis strains


0 0.02 0.04 0.06 0.08 0.1

17 days 19 days 23 days 25 days The

rat

io o

f nat

ive

redu

ced

glut

athi

one

in to

tal

glut

athi

one

The ratio of native GSH to total glutathion in two K. brevis strains


106

the presence of brevetoxin in K. brevis affects the amount of thiol and selenol groups

in cells.

Figure 60 The fluorescence is released by the reaction of sel-green probe with thiol groups and selenol groups in K. brevis homogenate. The homogenate (80 µl, 0.8 mg / ml of protein) mixed with sel- green (20 µl, 20 µM). A control sample was prepared by the addition of an equal volume of buffer instead of homogenate. The fluorescence was normalized to protein concentration in the homogenate 4.3.4 The activity of Trx and Grx in two K. brevis strains

The activities of Trx and Grx in K. brevis homogenates were determined using

fluorescent activity assay kits according to the manaufacturer’s (Cayman Chemical)

instructions. The reactions catalyzed by these enzymes and their mechanisms were

described in Chapter 3. A known concentration Trx (20 nM) was served as control.

Although same amount of homogenate was used to detect the low toxic strain showed

higher activity of Trx and Grx comparing to high toxic strain (Figure 61 and 62). It

had been shown that 3-fold activity of Trx and 1.7-fold activity of Grx was detected

in low toxic strain compaing to high toxic strain. However, the fluorescence caused

by two homogenates is much less than known-concentrtion Trx. Thus the results are

not persuasive. Increasing the amount of homogenate should be considered in the

further studying.

0

1

2

3

0 20 40 60 80 100 120 140 Fluo

resc

ence

/ pr

otei

n (µ

g)

Time (min)

The reaction of sel-green probe with K. brevis homogenate

high toxic strain low toxic strain

107

Figure 61 Using the fluorescent Trx / TrxR system kit detects the Trx activity in K. brevis homogenate. The homogenate (30 µg of protein) was incubated with NADPH (17.5 µl), TrxR (35 µl, 1 µM) and assay buffer (113 µl) for 30 min at 37 0C. Fluorescent substrate (70 µl) was added in each tube

Figure 62 Using the fluorescent Grx kit detects the Grx activity in K. brevis homogenate. The homogenate (30 µg of protein) was mixed with NADPH (10 µl), reduced glutathione (10 µl, 0.1 M), GR (10 µl, 10 µM), assay buffer (200 µl) and stabilized reagent and fluorescent substrate

4.4 Conclusion

Because of the discoveries found in chapter 2 and 3, we determined that the two K.

brevis strains not only differ in the amount of toxin but also in NPQ performance and

ROS level. Indeed, the low toxic strain cannot perform NPQ whereas high toxic strain

does. In addition, the low toxic strain produces more ROS than high toxic strain

(Table 11).

6000 8000

10000 12000 14000 16000 18000

0 20 40 60 80 100

Fluo

resc

ence

Time (min)

Trx activity in K. brevis homogenate

high toxic strain low toxic strain control (20 nM hTrx)

10000 12000 14000 16000 18000 20000 22000

0 20 40 60 80

Fluo

resc

ence

Time (min)

Gultaredoxin acticvty in K. brevis homogenate high toxic strain low toxic strain control (1.5 nM grx)

108

Table 11 Comparison of NPQ and ROS in two K. brevis strains

NPQ Relative rate of ROS High toxic strain able 21.9 Low toxic strain disable 12.16

The modified papain assay revealed that the low toxic strain of K. brevis has twice

thiol content when it compared to the high toxic strain. The free thiol was monitored

via the Ellman’s assay over the lifetime of the cultures, which revealed that low toxic

strain has twice amount of reduced thiol than high toxic strains from 17 to 19 growing

days. This is also consistent with the previous modified papain assay. Additionally, on

selected days during the log phase of the living cells’ growth cycle, GR that could

reduce any oxidized GSSG to the GSH, was added to parallel samples to quantify

total glutathione and asses the ratio of reduced GSH to total GSH. This showed that

the high toxic strain and low toxic strain had similar total GSH when the cells in the

log phase of growth. However, the low toxic strain has a maximum 2.4-fold ratio of

reduced GSH to total GSH than high toxic strain. In addition to thiols, antioxidant

capacity and ascorbic acid content are not the same in the high toxic and low toxic

strains. At 19th growing days, the high toxic strain has 1.16-fold antioxidant capacity

than low toxic strain. However, 4.5-fold less of ascorbic acid was present in high

toxic strain than low toxic strain, which matched the ratio of native GSH to total GSH

(Table 12). Because in cells GSH can reduce dehydroascorbic acid to ascorbic acid.

Table 12 Comparison of various biochemical parameters in two K. brevis strains at 19th days

Thiol content

Ratio of native GSH to total GSH

Ascorbic acid Antioxidant capacity

High toxic strain 1 1 1 1.16-fold

Low toxic strain 1.97-fold 2.2-fold 4.5-fold 1

109

Taking the results from various assays under consideration, we do find a difference of

redox state, antioxidant capacity, GSH and ascorbic acid content between high and

low toxin-producing strains. It has been determined that TrxR is related to the

production of GSH and ascorbic acid, such as the reduction of dehydroascorbic acid

to ascorbic acid by TrxR. In addition, the discoveries found in chapter 3 reveal that

brevetoxin impacts the activity of Trx/TrxR system, which is the major reductive

system in the cell. Experimental evidence indicates that the effect is caused by the

alkylation of TrxR by brevetoxin. Certain antioxidants such as thiols and ascorbic

acid are negatively correlated with brevetoxin content in K. brevis. These discoveries

provide evidence to support the hypothesis that brevetoixn impacts the reductive

system in cells through reaction with TrxR or other thiol containing proteins. Also,

the low toxin-producing strain has higher level of ROS and ascorbic acid than high

toxin-producing, which is able to support the difference at NPQ performance and

ROS level in two strains. Although it seems that total antioxidant capacity is in

conflict with our expectations, antioxidant capacity includes many enzymatic or non-

enzymatic systems, such as the TrxR/Trx and GSH systems, carotenoids, vitamin E

and so on. It is likely that brevetoxin deficiency or the reaction of brevetoxin with

proteins is the factor that causes the different performance or capacity in K. brevis

cells. But it still need further study to explore.

Furthermore, it is likely that those discoveries perhaps create a new horizon to

understand the role of brevetoxin in K. brevis or the impacts of damage caused by

brevetoxin.

110

4.5 Materials and Methods

4.5.1 Culture methods

Method: K. brevis culture (Wilson strains) were obtained from Mote Marine

Laboratory (Sarasota, Florida) and maintained in L1-Si medium, with the exception

that the NH 15 vitamin supplement (Gates and Wilson 1960) replaced the L-1

supplement, in a growth chamber at ~20ºC under 35 µmol.photons.m-2.s-1. Growth

was monitored by counting a 1:10 dilution of culture in Z pak reagent using a

Beckman Z-series Coulter Counter with aperture size between 10~30 µm according to

the manufacturer’s instructions.

4.5.2 Preparation of K. brevis homogenate

Method K. brevis culture (200–500 ml) was concentrated by centrifugation (5 min at

466 X g) and the supernatant was discarded. The cells were resuspended in

phosphate-buffered saline (PBS) and vortexed for 1 min. This suspension was

centrifuged (10 min at 14,000 X g) to remove cellular debris and the supernatant was

transferred to a clean tube. An aliquot (50 µl) was reserved for a protein assay.

Samples were quantitated against BSA standards (0, 1, 0.5, 0.25, 0.125, and 0.0625

mg /ml) prepared in PBS using the Coomassie Protein Assay Reagent (BioRad)

according to the manufacturer’s instructions. Final protein concentrations ranged from

0.2–1 mg / ml

4.5.3 ABTS assay

The materials for ABTS assay ABTS (2,2-azinobis (3-ethenelbenzothiazoline-6-

sulfonic acid)), (NH4)2S2O8, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-

carboxylic acid), ascorbic acid oxidase. High and low toxic K. brevis strain

111

homogenate was prepared through the pervious method. UV/Visible measurements

were performed in 96 or 384 well microplates using a Synergy® 2 (Biotek Instrument,

Inc.)

The method for ABTS assay Total antioxidant capacity of K. brevis homogenate

was determined by 2,2-azinobis (3-ethenelbenzothiazoline-6-sulfonic acid) radical

cation (ABTS.+) decolorization and expressed as trolox equivalents. ABTS.+ was

produced by oxidation of aqueous ABTS (1.3 mL, 7 mM) with ammonium persulfate

(21.2 µl, 2.45 mM). The mixture was allowed to stand at room temperature in the

dark for 12-16 h before use. The ABTS.+ solution was diluted with PBS to an

absorbance of 0.7. Either 50 ul K. brevis homogenate or trolox standards (0 µM, 25

µM, 50 µM, 100µM, 200 µM) were added to the ABTS.+ solution (100 µl). The

absorbance was read at 734 nM after incubation for 5 min. Ascorbic acid

concentration (as trolox equivalents) of K. brevis homogenate was estimated by

incubating with of ascorbic acid oxidase (2 µl, 0.01 U/ µL) for 30 min prior to the

assay and comparing absorbance to parallel samples.


Materials for modified papain assay: Papain, methyl methanethiosulfonate, N-

benzoyl-L-arginine-p-nitroanilide and cystamine were purchased from Thermo

Scientific Co.. High and low toxic K. brevis strain homogenate was prepared through

the pervious method. UV/Visible measurements were performed in 96 or 384 well

microplates using a Synergy® 2 (Biotek Instrument, Inc.)

112

Preparation of papain-S-SCH3 (~1.2 mg / ml)1.5 ml of papain stock solution (26.4

mg / ml) was added to a solution of cysteine (0.6 mM, 28.5 ml of 20 mM sodium

phosphate – 1 mM EDTA buffer). The solution was kept at room temperature for 30

min. The methyl methanethiosulfonate (117 µmol) was added in mixture. The

reaction mixture was kept in ice for 4 hr. The mixture was dialyzed (Mr cutoff 6000-

8000) against 5 mM sodium acetate buffer, containing 50 mM NaCl, pH 4.7 at 4oC 12

hr.

Method for modified papain assay: The culture was treated under dark adapt or sun

light for 1 hr before making homogenate. Papain-S-SCH3 solution (0.5 ml, 0.6 mg /

ml), 0.7 ml substrate and 15 µl cystamine were added in one test tube which contained

200 µl supernatant. A tube containing papain-S-SCH3, without homogenate or

substrate, serves as control or background. The mixture was carried out in triplicate in

plate. Each well was added 300 µl mixture. Record the absorbance at 410 nm

(Synergy™ 2, Bioteck Instrument, Inc.) every 60 min up to 15 hr.


Materials for Ellman’s assay: DTNB and cysteine was purchased from Thermo

Scientific Co.. High and low toxic K. brevis strain homogenate was prepared through

the pervious method. Glutathione reductase was from Grx kit purchased from

Cayman Chemical. UV/Visible measurements were performed in 96 or 384 well

microplates using a Synergy® 2 (Biotek Instrument, Inc.)

Method for Ellman’s assay: K. brevis homogenates (300 µl, 0.3-0.5 mg / ml protein)

was mixed with reaction buffer (700 µl, 0.1 M sodium phosphate, 1 mM EDTA, pH

113

8.0) and Ellman’s reagent (20 µl, 4 mg / ml). After incubation for 15 min at room

temperature absorbance was read at 410 nm (Synergy™ 2, Bioteck Instrument, Inc).

Homogenate prepared similarly, but without Ellman’s reagent was used to determine

background absorbance which was subtracted from each sample. Ellman’s reagent in

reaction buffer served as the control. Samples were quantitated against cysteine

standards (0, 0.3, 0.6 and 1.2 mM) prepared in reaction buffer in place of homogenate

by using the Ellman’s reagent according to the manufacturer’s instructions (Thermo

Fisher). Samples and cysteine standards were analyzed in triplicate. The amount of

thiol in homogenate was normalized to protein concentration in the homogenate.

Ellman’s assay of two strains with GR K. brevis homogenates (275 µl, 0.3-0.5 mg /

ml) was mixed with NADPH (100 µl, 1 mg / ml), glutathione reductase (3 µl, 10 µM)

and Ellman’s reagent (25 µl, 4 mg / ml). Immediately the absorbance was read at 410

nm at room temperature up to 30 min. Homogenate prepared similarly, but without

Ellman’s reagent was used to determine background absorbance which was subtracted

from each sample. Ellman’s reagent in reaction buffer served as the control. Samples

were quantitated against cysteine standards (0, 0.3, 0.6 and 1.2 mM) prepared in

reaction buffer in place of homogenate by using the Ellman’s reagent. Samples and

cysteine standards were analyzed in triplicate.

4.5.6 Sel- green reacts with homogenate of tow K. brevis strains

Materials: Sel-Green was synthesized by Freddy Rodriguez. High and low toxic K.

brevis strain homogenate was prepared through the pervious method. UV/Visible

measurements were performed in 96 or 384 well microplates using a Synergy® 2

(Biotek Instrument, Inc.)

114

Method: The selenol selective probe, Sel-green, was synthesized according to

published methods (Zhang, Ge et al. 2015). The homogenate was made resulting in

0.8 mg / ml of protein both in high toxic and low toxic homogenate. Sel- green (20 µl,

20 µM) was mixed with high toxic strain or low toxic strain homogenate (80 µl).

Samples were prepared in triplicate. A control sample was prepared by the addition of

an equal volume of buffer instead of homogenate. A blank sample was prepared

without Sel-green. Fluorescence at 502 (λex = 370 nm) was monitored for 120 mins.

4.5.7 Thioredoxin activity in homogenate of two strains K. brevis

Materials: Enzyme assay kit was purchased from Cayman Chemical. Assay kit was

used according to the manufacturer’s instructions with noted exceptions. High and

low toxic K. brevis strain homogenate was prepared through the pervious method.

Fluorescence measurements were performed in 96 or 384 well microplates using an

Infinite® M1000 PRO (Tecan Group Ltd.) microplate readers.

Method: The homogenate was made resulting in 1.0 mg / ml and 1.5 mg / ml of

protein high toxic and low toxic homogenate. Assay buffer (113 µl, 0.2 mg / ml

bovine serum albumin in 50 mM Tris-HCl and 1 mM EDTA, pH 7.5) was mixed with

17.5 µl NADPH in one clean tube. Thioredoxin reductase (35 µl, 1 µM) was added

into the mixture. The homogenate was incubated with the mixture for 30 min at 37 0C.

70 µl fluorescent substrate was added in each tube. The mixture was carried out in

triplicate in plate. Each well is added 100 µl mixture. Record its fluorescence at the

emission / excitation at 545 nm / 520 nm every 10 min for 3 hr.

115

4.5.8 Grx activity in homogenate of two strains K. brevis

Materials: Fluorescent Grx kit was purchase from Cayman Chemical. High and low

toxic K. brevis strain homogenate was prepared as mentioned above. Fluorescence

measurements were performed in 96 or 384 well microplates using an Infinite®

M1000 PRO (Tecan Group Ltd.) microplate readers.

Method: The homogenate was made resulting in 1.0 mg / ml and 1.5 mg / ml of

protein high toxic and low toxic homogenate. Assay buffer (200 µl) was mixed with

reduced glutathione (10 µl, 0.1 M) in one clean tube. NADPH (10 µl) and stabilized

reagent were added with mixture. Glutathione reductase (10 µl, 10 µM) was applied

into the mixture. The distilled water was added it up to 1 ml. The homogenate and

fluorescent substrate were added with mixture in each tube. Immediately the mixture

was carried out in triplicate in plate. Each well was added 100 µl mixture. Record its

fluorescence at the emission / excitation at 545 nm / 520 nm every 10 min for 3 hr.

116

Chapter 5 Summary and future work

5.1 Summary

This dissertation describes efforts to understand the principle role that brevetoxin

plays in K. brevis. The dissertation includes four components: i) localization of

brevetoxin in K. brevis cells ii) identification of the target proteins of brevetoxin iii)

characterization of the interaction between a target protein and brevetoxin iv)

comparison of the various metabolic and biochemical parameters in two strains of K.

brevis having different toxin content.

Brevetoxin was localized to the chloroplast of live K. brevis cells through the

fluorescently labeled brevetoxin probe. Using a brevetoxin photoaffinity probe, two

native receptors of brevetoxin were discovered from K. brevis. Based on the BLAST

analysis, LHC II and Trx were identified as binding partners for brevetoxin in K.

brevis. An interaction between brevetoxin and a transmembrane target protein, LHC

II, was discovered, which is related to the NPQ mechanism to protect the cells from

damage by ROS. Also, PbTx-2 appeared to inhibit the Trx/TrxR system. However,

PbTx-2 promoted the reduction of DTNB by TrxR. Although there is no direct

evidence to prove PbTx-2 reacts with mammalian TrxR by alkylation of the α, β-

unsaturated aldehyde of brevetoxin with selenocysteine in TrxR, several pieces of

indirect evidence supported this conclusion. Because of this deactivation, mammalian

TrxR could not reduce the Trx, and thus, the insulin disulfide bonds could not be

reduced by Trx either. However, the reaction center on N-terminal of mammalian

TrxR still can reduce some small molecule such as DTNB.

Thus, according to the identification of the two native receptors and interaction

between brevetoxin with mammalian Trx/TrxR system, follows that the redox state

117

and antioxidant capacity of K. brevis may be related to toxin content. It had been

revealed that high toxic strain could perform NPQ whereas low toxic strain could not

perform NPQ. The low toxic strain had higher levels of ROS than the high toxic strain.

The modified papain assay, Ellman’s assay, and ABTS assay were used to compare

the free thiol groups and antioxidants in the high and low toxic strains. In general, the

high toxic strain had a higher antioxidant capacity than the low toxic strain. However,

the low toxic strain consistently exhibited higher levels of free thiol groups and

ascorbic acid. Both of these would be influenced by the TrxR/Trx activity in the cells.

The low toxic strain has slightly higher activity of Trx system and Grx compared to

high toxic strain. Therefore, brevetoxin content seems to be correlated with redox

homeostasis of the cells.

It is likely that brevetoxins plays an important role in K. brevis. It is highly possible

that brevetoxin affects the reductive system in cells, leading to the impact on various

metabolic and biochemical parameters in two strains of K. brevis. In order to better

understand these phenomenons, additional experiments are still needed to be done to

prove our hypothesis.

5.2 Future work

5.2.1 Identify all biological receptors for brevetoxin in K. brevis and localize the

binding site on native receptors

Our receptor binding experiments at pH 7 have consistently shown one additional

high MW bands between 57 and 75 kD (Figure 63). A third band at 37 kD has been

observed sporadically. These two bands have been excised from gels and can be sent

immediately to the mass spectrometry facility at the University of Massachusetts at

118

Amherst to be identified.

Figure 63 Spinach homogenate was incubated with photoaffinity probe in PBS (pH=7) for 1 hour. Lane 1: Molecular Weight markers Lane 2: The first elution. Lane3: The second elution. Lane4: Total protein. Because the increase effect of TrxR by PbTx-3 was observed as well in DTNB assay.

Also, PbTx-3 and PbTx-2 both have an aldehyde at A-ring. We suspect PbTx-3 may

bind or react with Trx or TrxR enzyme as well. Perhaps, the A-ring is another active

site for brevetoxin B. Thus, a brevetoxin (PbTx-2 or PbTx-3) photoaffinity probe

(Figure 64) which includes a photoactive diazirine to interact with its receptor can be

synthesized to incubate with K. brevis homogenate. This time, the ligand will be

introduced A-ring position in PbTx-2 or PbTx-3. The photolysis, purification and

identification are same as previous experiment. We may or may not discover new

target proteins.

Figure 64 A-ring derivative from PbTx-2 or PbTx-3 A-ring diol If in order to identify the specific binding amino acid in target protein, the same

photoaffinity probe (K-ring derivative or A- ring derivative) is incubated with K.

O

CF3

N

N

3

O

O

MeO

HOH

H

B

119

brevis homogenate. After photolysis, the probe may react to form a covalent cross-

link to a variety of amino acid residues within the target protein. This time, the protein

sample will be digested by trypsin to its peptide constituents before purification. The

same procedure will be used to isolate the full protein by passing the digest through a

strepavadin column. The target fragment, linked with biotin will be eluted from the

streptavidin column by heat treatment. SDS-PAGE will be performed to analyze the

captured fragments. Finally, send the bands to the mass spectrometry facility at the

University of Massachusetts at Amherst.

5.2.2 Analysis of the essential proteins, carotenoids and lipids for NPQ in low vs

high toxin K. brevis

The low toxic strain does not perform NPQ. It is still unclear if the inability to

perform NPQ is due to the absence of brevetoxin, or if the lack of brevetoxin is a

result of the NPQ deficiency. It is necessary to determine if the low toxic strain is

deficient in one or more of the essential components of NPQ: zeaxanthin and

violaxanthin.

Quantitative polymerase chain reaction (qPCR) can be used to amplify and quantify

the VDE and ZE mRNA in low and high toxic strains. If we can determine the RNA

of VDE and ZE in two strains, we will do further study at translational level. Thus,

western blot analysis can be used in further studies to compare protein extracts of the

low and high toxic strains of K. brevis using three different antibodies: LHC II, VDE

and ZE. These three antibodies can be purchased from Swedish company Agrisera.

Hopefully it is possible to determine if one or more of these essential components is

absent or reduced in the low-toxin culture.

120

5.2.3 Determination the role of brevetoxin in two K. brevis strains

Although the difference of NPQ, ROS, antioxidant capacity and biochemical

parameters in high toxin and low toxin producing strains of K. brevis were determined,

the reasons caused this phenomenon were still unknown. Does brevetoxin induce

those difference? In order to understand it, my idea is adding PbTx-2 with low toxic

strain for 25 days. The cells are used unused medium to wash at least twice. The

homogenate is made as previous method. DTNB assay, ROS and ABTS assay can be

applied again to compare the difference between high toxic strain and low toxic strain

which is treated with PbTx-2. The difference becomes smaller or totally disappear,

which means brevetoxin is one of the reasons that cause the distinction between high

and low toxic strains.

121

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VITA

WEI CHEN

EDUCATION

2007 - 2011 B.S., Chemistry

Wuhan institute of technology, Wuhan, China

2011 - 2016 Doctoral Candidate

Florida International University, Miami, Florida, USA

PUBLICATIONS and PRESENTATIONS

*Cassell, R. T., *W. Chen, S. Thomas, L. Liu and K. S. Rein “Brevetoxin, the dinoflagellate neurotoxin, localizes to thylakoid membranes and interacts with the light-harvesting complex II (LHCII) of photosystem II.” ChemBioChem Volume 16, Issue 7, pages 1060–1067, May 4, 2015 (* represents the equal contribution)

W. Chen, F. Rodriguez del Rey, T. Anupama, C. E. Zavala and K. S. Rein “Brevetoxin-2, inhibits the C-terminal redox center of mammalian thioredoxin reductase-1: Implications for a new mechanism of toxicity.” Chemical Research in Toxicology (in- progress)

W. Chen, F. Rodriguez del Rey, R. Colon, M. Durham and K. S. Rein “Brevetoxin-2, is an Inhibitor of thioredoxin reductase: lmplcations for endogenous function and toxicity” Harmful Algae (in- progress)

W. Chen and K. S. Rein “The inhibition of the mammalian thioredoxin / thioredoxin reductase system by brevetoxins” 2016 Florida Annual Meeting and Exposition of the Florida Section of the American Chemical Society, Tampa, FL 05/06-05/08 (Oral Presentation)

W. Chen, F. Rodriguez and K. S. Rein “Characterizing the redox status of high and low toxin producing K. brevis strains” 2016 Florida Annual Meeting and Exposition of the Florida Section of the American Chemical Society, Tampa, FL 05/06-05/08 (Poster Presentation) W. Chen and K. S. Rein “The interaction between the dinoflagellate neurotoxin and the thioredoxin system” 2015 8th Symposia of Harmful Algae in the U.S., Long beach, CA, 11/15-11/19. (Oral Presentation) W. Chen and K. S. Rein “The interaction between the dinoflagellate neurotoxin and the thioredoxin system” 2015 8th Symposia of Harmful Algae in the U.S., Long beach, CA, 11/15-11/19. (Poster Presentation) W. Chen and K. S. Rein “The interaction between the dinoflagellate neurotoxin and its native receptors” 2015 Florida Annual Meeting and Exposition of the Florida Section of the American Chemical Society, Tampa, FL, 05/08-05/10. (Oral Presentation)

W. Chen, Cassell, R. T. and K. S. Rein “Localiaztion of brevetoxins and photoaffinity labeling of target proteins” 2013 7th Symposia of Harmful Algae in the U.S. Sarasota, FL, 10/27-10/31 (Poster Presentation)

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T. Anupama, W. Chen, S. Rolle, Y. Liu and K. S. Rein “The effect of trolox on brevetoxicosis in human lymphoblast cells” 2016 XII congress of the pan American section of the international society of toxinology, Miami beach, FL, 09/18-09/23 (Poster presentation)

characterization of interaction between brevetoxin and its

Documents